Formulations of single domain antigen binding molecules

ABSTRACT

The invention relates to formulations of single domain antigen binding molecules, e.g., nanobody molecules, in particular formulations of TNF-binding nanobody molecules. The single domain antigen binding molecules can include one or more single binding domains that interact with, e.g., bind to, one or more target proteins. The formulations are useful, e.g., as pharmaceutical formulations. Method of preparing, and using the formulations described herein, to treat, e.g., TNF-associated disorders, are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 61/109,474, filed onOct. 29, 2008, the entire contents of which are hereby incorporated byreference in their entirety. This application also incorporates byreference the International Application filed with the U.S. ReceivingOffice on Oct. 29, 2009, entitled “Formulations of Single Domain AntigenBinding Molecules” and bearing attorney docket number W2023-7039WO.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Oct. 27, 2009, is named982855_(—)1.txt, and is 6,456 bytes in size.

BACKGROUND

Advances in biotechnology have made it possible to produce a variety ofproteins for pharmaceutical applications using recombinant DNAtechniques. Because proteins tend to be larger and more complex thantraditional organic and inorganic drugs, the formulation of suchproteins poses special problems. For a protein to remain biologicallyactive, a formulation must preserve the conformational integrity of atleast a core sequence of the protein's amino acids, while at the sametime protecting the protein's multiple functional groups fromdegradation. Degradation pathways for proteins can involve chemicalinstability (i.e. any process which involves modification of the proteinby bond formation or cleavage resulting in a new chemical entity) orphysical instability (i.e. changes in the higher order structure of theprotein). Chemical instability can result from, for example,deamidation, racemization, hydrolysis, oxidation, beta elimination ordisulfide exchange. Physical instability can result from, for example,denaturation, aggregation, precipitation or adsorption. Three commonprotein degradation pathways are protein aggregation, deamidation andoxidation (Cleland et al. Critical Reviews in Therapeutic Drug CarrierSystems 10(4): 307-377 (1993)).

Freeze-drying is a commonly employed technique for preserving proteinswhich serves to remove water from the protein preparation of interest.Freeze-drying, or lyophilization, is a process by which the material tobe dried is first frozen and then the ice or frozen solvent is removedby sublimation in a vacuum environment. An excipient may be included inpre-lyophilized formulations to enhance stability during thefreeze-drying process and/or to improve stability of the lyophilizedproduct upon storage (Pikal, M. Biopharm. 3(9)26-30 (1990) and Arakawaet al. Pharm. Res. 8(3):285-291 (1991)).

Therefore, the need still exists for developing protein formulations,particularly for subcutaneous administration, that are stable forlong-term storage and delivery.

SUMMARY

The invention relates to formulations of single domain antigen bindingmolecules (also referred to herein as “SDAB molecules” (e.g., nanobodymolecules, in particular formulations of TNF-binding nanobodymolecules). The SDAB molecule can include one or more single antigenbinding domains that interact with, e.g., bind to, one or more targetproteins. The formulations are useful, e.g., as pharmaceuticalformulations, for administration to a subject, e.g., a human. Method ofpreparing, and using the formulations described herein, to treat orprevent, e.g., TNF-associated disorders, are also disclosed.

[Note: Nanobody™ and Nanobodies™ are registered trademarks of AblynxN.V.]

Accordingly, in one aspect, the invention features a formulation thatincludes (a) an SDAB molecule, e.g., a nanobody molecule (e.g., aTNF-binding nanobody molecule); (b) a lyoprotectant; (c) (optionally) asurfactant; (d) (optionally) a bulking agent; (e) (optionally) atonicity adjusting agent; (f) (optionally) a stabilizer; (g)(optionally) a preservative, and (h) a buffer, such that the pH of theformulation is about 5.0 to 7.5. In some embodiments, the formulation isa liquid formulation, a lyophilized formulation, a reconstitutedlyophilized formulation, an aerosol formulation, or a bulk storageformulation (e.g., frozen bulk storage formulation). In certainembodiments, the formulation is administered to a subject by injection(e.g., subcutaneous, intravascular, intramuscular or intraperitoneal) orby inhalation.

In certain embodiments, the SDAB molecule, e.g., the nanobody molecule(e.g., the TNF-binding nanobody molecule), in the formulation is at aconcentration of about 0.5 mg/mL to about 350 mg/mL, about 0.5 mg/mL toabout 300 mg/mL, about 0.5 mg/mL to about 250 mg/mL, about 0.5 mg/mL toabout 150 mg/mL, about 1 mg/ml to about 130 mg/mL, about 10 mg/ml toabout 130 mg/mL, about 50 mg/ml to about 120 mg/mL, about 80 mg/ml toabout 120 mg/mL, about 88 mg/ml to about 100 mg/mL or about 10 mg/ml,about 25 mg/ml, about 50 mg/ml, about 80 mg/ml, about 100 mg/mL, about130 mg/ml, about 150 mg/ml, about 200 mg/ml, about 250 mg/ml or about300 mg/ml.

In other embodiments, the lyoprotectant of the formulation is a sugar,e.g., sucrose, sorbitol, or trehalose. For example, the lyoprotectantcan be sucrose, sorbitol, or trehalose at a concentration about 2.5% toabout 10%, about 5% to about 10%, about 5% to about 8%, or about 4%,about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about7.5%, about 8%, about 8.5%, or about 9% (weight/volume).

In yet other embodiments, the buffer in the formulation is a histidinebuffer at a concentration about 5 mM to about 50 mM, about 5 mM to about40 mM, about 5 mM to about 30 mM, about 10 mM to about 20 mM, or about10 mM, about 20 mM, or about 30 mM. In other embodiments, the buffer inthe formulation is a Tris buffer present at a concentration of less thanabout 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM toabout 30 mM, about 10 mM to about 20 mM, or about 10 mM, about 20 mM, orabout 30 mM. The pH of the buffers of the formulation is generallybetween about 5 and 7. In some specific embodiments, the pH of thebuffer of the formulation is about 5 to about 7.5, about 5.5 to about7.2. For example, the pH of the buffer can be about 5, 5.5, 5.8-6.1, 6,6.1, 6.5 or 7.

In some embodiments, the formulation (optionally) includes a surfactantat a concentration of about 0.001% to 0.6%, e.g., about 0.01% to 0.6%,about 0.1% to 0.6%, about 0.1% to 0.5%, about 0.1% to 0.4%, about 0.1%to 0.3%, about 0.1% to 0.2%, or about 0.01% to 0.02%. In some cases, theformulation contains greater than 0% and up to about 0.6% (e.g., about0.1% to 0.2% of polysorbate-20, polysorbate-40, polysorbate-60,polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitanmonolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitanmonooleate, sorbitan trilaurate, sorbitan tristearate, sorbitantrioleaste, or a combination thereof. In specific embodiments, theformulation contains about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%,0.006%, 0.007%, 0.008%, 0.009%, 0.01% to 0.02%, 0.01%, 0.02%, 0.03%,0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.1% to 0.2%, 0.11%,0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19% or 0.2% ofpolysorbate-80. Alternatively, the formulation can include poloxamer-188at about 0.01% to 0.6%, about 0.1% to 0.6%, about 0.1% to 0.5%, about0.1% to 0.4%, about 0.1% to 0.3%, or about 0.1% to 0.2%.

In certain embodiments, the formulation (optionally) includes a bulkingagent, e.g., glycine, at a concentration from about 10 to about 200 mM,from about 25 to about 175 mM, from about 50 to about 150 mM, from about75 to about 125 mM, or about 100 mM.

In other embodiments, the formulation (optionally) further includes atonicity adjusting agent, e.g., a molecule that renders the formulationsubstantially isotonic or isoosmotic with human blood. Exemplarytonicity adjusting agents include sucrose, sorbitol, glycine,methionine, mannitol, dextrose, inositol, sodium chloride, arginine andarginine hydrochloride.

In yet other embodiments, the formulation (optionally) additionallyincludes a stabilizer, e.g., a molecule which, when combined with aprotein of interest (e.g., the SDAB molecule) substantially prevents orreduces chemical and/or physical instability of the protein of interestin lyophilized, liquid or storage form. Exemplary stabilizers includesucrose, sorbitol, glycine, inositol, sodium chloride, methionine,arginine, and arginine hydrochloride. In certain embodiments, theformulation includes a stabilizer in one or more of the followingranges: Sucrose from about 1% to about 12% (e.g., about 5%, about 7.5%,about 8% or about 10%); sorbitol from about 1% to about 7% (e.g., about3%, about 4%, about 5%); inositol from about 1% to about 5%; glycinefrom about 10 mM to about 125 mM (e.g., about 25 mM to 100 mM, about 80mM, about 90 mM, or about 100 mM); sodium chloride from about 10 mM to150 mM (e.g., about 25 mM to 100 mM, about 55 mM); methionine from about10 mM to about 100 mM (e.g., about 10 mM, about 20 mM, about 100 mM);arginine from about 10 mM to about 125 mM (e.g., about 25 mM to about120 mM, or about 100 mM); arginine hydrochloride from about 10 mM toabout 70 mM (e.g., about 10 mM to about 65 mM, or about 55 mM).

In other embodiments, the formulation may further include methionine, ata concentration from about 10 to about 200 mM, from about 25 to about175 mM, from about 50 to about 150 mM, from about 75 to about 125 mM, orabout 100 mM.

In one embodiment, a component of the formulation can function as one ormore of a lyoprotectant, a tonicity adjusting agent and/or a stabilizer.For example, depending on the concentration of a component, e.g.,sucrose, it can serve as one or more of a lyoprotectant, a tonicityadjusting agent and/or a stabilizer. In other embodiments where severalof the components are required in a formulation, different componentsare used. For example, where the formulation requires a lyoprotectant, atonicity adjusting agent and a stabilizer, different components are used(e.g., sucrose, glycine and inositol can be used in combinationresulting in a combination of a lyoprotectant, a tonicity adjustingagent and a stabilizer, respectively).

In one embodiment, the formulation includes (a) an SDAB molecule, e.g.,a nanobody molecule (e.g., a TNF-binding nanobody molecule) at aconcentration of about 0.5 to about 300 mg/mL, e.g., at about 1 mg/mL,about 10 mg/mL, about 25 mg/mL, about 50 mg/mL, about 80 mg/mL, about 88mg/mL, about 100 mg/mL, about 118 mg/mL, about 130 mg/mL, about 150mg/mL, or about 250 mg/mL; (b) sucrose at a concentration of about 5% toabout 10%, e.g., about 5%, about 6%, about 6.5%, about 7%, about 7.5%,about 8%, about 10%; (c) polysorbate-80 at a concentration of about 0 toabout 0.6%, e.g., 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or0.6%; (d) (optionally) glycine at a concentration of about 0 to about100 mM, e.g., 100 mM; (e) (optionally) methionine at a concentration ofabout 0 to about 100 mM, e.g., 100 mM; and (f) a histidine buffer (at aconcentration about 10 mM to about 20 mM) or a Tris buffer (at aconcentration about 20 mM), such that the pH of the formulation is about5.0 to 7.5, e.g., 5, 5.5, 5.8-6.1, 6, 6.1, 6.5, or 7.

In one embodiment, the formulation is a liquid formulation. In onerepresentative embodiment, the liquid formulation includes a) an SDABmolecule, e.g., a nanobody molecule (e.g., a TNF-binding nanobodymolecule) at a concentration of about 10 to about 150 mg/mL, e.g., about25 mg/mL, about 50 mg/mL, about 80 mg/mL, about 88 mg/mL, about 100mg/mL, about 118 mg/mL, about 130 mg/mL; (b) sucrose at a concentrationof about 5% to about 10%, e.g, about 7% to about 8%, e.g., 7.5%; orsorbitol from about 1% to about 7% (e.g., about 3%, about 4%, about 5%)(c) polysorbate-80 at a concentration of about, e.g., about 0.01% to0.02% (e.g., 0.01%); (d) (optionally) glycine at a concentration ofabout 0 to about 100 mM, e.g., 100 mM; (e) (optionally) methionine at aconcentration of about 0 to about 100 mM, e.g., 100 mM; and (f) ahistidine buffer (at a concentration about 10 mM to about 20 mM), or aTris buffer (at a concentration about 20 mM), such that the pH of theformulation is about 5 to 7.5, e.g., 5, 5.5, 5.8-6.1, 6, 6.1, 6.5, or 7.The liquid formulation can be present in an article of manufacture, suchas a device, a syringe or a vial with instructions for use. In certainembodiments, the syringe or a vial is composed of glass, plastic, or apolymeric material, such as cyclic olefin polymer or copolymer. In otherembodiments, the formulation can be present in an injectable device(e.g., an injectable syringe, e.g., a prefilled injectable syringe). Thesyringe may be adapted for individual administration, e.g., as a singlevial system including an autoinjector (e.g., a pen-injector device),and/or instructions for use. The formulation can be administered to asubject, e.g., a patient, by in injection, e.g., peripheraladministration (e.g., subcutaneous, intravascular, intramuscular orintraperitoneal administration).

In other embodiments, the formulation is a lyophilized formulation. Inone representative embodiment, the lyophilized formulation includes a)an SDAB molecule, e.g., a nanobody molecule (e.g., a TNF-bindingnanobody molecule) at a concentration of about 10 to about 150 mg/mL,e.g., about 25 mg/mL, about 50 mg/mL, about 80 mg/mL, about 88 mg/mL,about 100 mg/mL, about 118 mg/mL, about 130 mg/mL; (b) sucrose at aconcentration of about 5% to about 10%, e.g, about 4% to about 7%, e.g.,5%; (c) polysorbate-80 at a concentration of about, e.g., 0.01% to 0.02%(e.g., 0.01%); (d) (optionally) glycine at a concentration of about 0 toabout 100 mM, e.g., 100 mM; (e) (optionally) methionine at aconcentration of about 0 to about 100 mM, e.g., 100 mM; and (f) ahistidine buffer (at a concentration about 10 mM to about 20 mM, e.g.,about 20 mM), or a Tris buffer (at a concentration about 20 mM), suchthat the pH of the formulation is about 5 to 7.5, e.g., 5, 5.5, 5.8-6.1,6, 6.1, 6.5 or 7. The lyophilized formulation can be reconstituted bymixing the lyophilate with a suitable aqueous composition.

In yet other embodiments, the formulation is a bulk storage formulation.In one representative embodiment, the bulk storage formulation includesa) an SDAB molecule, e.g., a nanobody molecule (e.g., a TNF-bindingnanobody molecule) at a concentration of about 80 mg/mL to 300 mg/ml,e.g., about 150 mg/mL, about 175 mg/mL, about 200 mg/mL, about 250mg/mL, about 275 mg/mL, or about 300 mg/mL; (b) sucrose at aconcentration of about 5% to about 10%, e.g, about 4% to about 8%, e.g.,5%, or 7.5%; (c) polysorbate-80 at a concentration of about, e.g., 0.01%to 0.02%; (d) (optionally) glycine at a concentration of about 0 toabout 100 mM, e.g., 100 mM; (e) (optionally) methionine at aconcentration of about 0 to about 100 mM, e.g., 100 mM; and (f) ahistidine buffer (at a concentration about 10 mM to about 20 mM) or aTris buffer (at a concentration about 20 mM), such that the pH of theformulation is about 5 to 7.5, e.g., 5, 5.5, 5.8-6.1, 6, 6.1, 6.5 or 7.The bulk storage formulation can be frozen. In certain embodiments, thebulk storage formulation can be prepared in large scale, e.g., greaterthan 10 liters, 50 liters, 100, 150, 200 or more liters.

In certain embodiments, the SDAB molecule, e.g., the nanobody molecule(e.g., the TNF-binding nanobody molecule) of the formulation includesone or more single binding domains (e.g., one or more nanobodies). Forexample, the nanobody molecule can comprise, or consist of, apolypeptide, e.g., a single chain polypeptide, comprising at least oneimmunoglobulin variable domain (including one, two or threecomplementarity determining regions (CDRs)). Examples of SDAB moleculesinclude molecules naturally devoid of light chains (e.g., VHH,nanobodies, or camelid derived antibodies). Such SDAB molecules can bederived or obtained from camelids such as camel, llama, dromedary,alpaca and guanaco. In other embodiments, the SDAB molecule may includesingle domain molecules including, but not limited to, othernaturally-occurring single domain molecules, such as shark single domainpolypeptides (IgNAR); and single domain scaffolds (e.g., fibronectinscaffolds). Single domain molecules may be derived from shark.

In one embodiment, the SDAB molecule of the formulation is a singlechain polypeptide comprised of one or more single domain molecules. Inembodiments, the nanobody molecule is monovalent or multivalent (e.g.,bivalent, trivalent, or tetravalent). In other embodiments, the nanobodymolecule is monospecific or multispecific (e.g., bispecific, trispecificor tetraspecific). The SDAB molecule may comprise one or more singledomain molecules that are recombinant, CDR-grafted, humanized,camelized, de-immunized, and/or in vitro generated (e.g., selected byphage display). For example, the SDAB molecule can be a single chainfusion polypeptide comprising one or more single domain molecules thatbind to one or more target antigens. Typically, the target antigen is amammalian, e.g., a human, protein. In certain embodiments, the SDABmolecule binds to a serum protein, e.g., a human serum proteins chosenfrom one or more of serum albumin (human serum albumin (HSA)), fibrin,fibrinogen, or transferrin.

In one exemplary embodiment, the SDAB molecule of the formulation is atrivalent, bispecific molecule composed of a single chain polypeptidefusion of two single domain molecules (e.g., two camelid variableregions) that bind to a target antigen, e.g., tumor necrosis factor α(TNF α), and one single domain molecule (e.g., a camelid variableregion) that binds to a serum protein, e.g., HSA. The single domainmolecules of the SDAB molecule can be arranged in the following orderfrom N— to C-terminus: TNFα-binding single domain molecule—HSA-bindingsingle domain molecule—TNFα binding single domain molecule. It will beappreciated that any order or combination of single domain moleculesagainst one or more targets can be formulated as described herein.

In one embodiment, the SDAB molecule of the formulation is referred toherein as “ATN-103,” comprises, or consists of, the amino acid sequenceshown in FIG. 30 (SEQ ID NO:1), or an amino acid sequence substantiallyidentical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%or more identical to, or having up to 20, 15, 10, 5, 4, 3, 2, 1 aminoacid changes (e.g., deletions, insertions or substitutions (e.g.,conservative substitutions) relative to the amino acid sequence shown inFIG. 30). Examples of additional trivalent, bispecific nanobodymolecules that can be formulated as described herein include TNF24,TNF25, TNF26, TNF27, TNF28, TNF60 and TNF62 disclosed in Table 29 of WO2006/122786.

In certain embodiments, at least one of the single domain molecule ofthe SDAB molecule of the formulation binds to TNFα includes one, two, orthree CDRs having the amino sequence: DYWMY (SEQ ID NO:2) (CDR1),EINTNGLITKYPDSVKG (SEQ ID NO:3) (CDR2) and/or SPSGFN (SEQ ID NO:4)(CDR3), or having a CDR that differs by fewer than 3, 2 or 1 amino acidsubstitutions (e.g., conservative substitutions) from one of said CDRs.In other embodiments, the single domain molecule comprises a variableregion having the amino acid sequence from about amino acids 1 to 115 ofFIG. 30, or an amino acid sequence substantially identical thereto(e.g., an amino acid sequence at least 85%, 90%, 95% or more identicalto, or having up to 20, 15, 10, 5, 4, 3, 2, 1 amino acid changes (e.g.,deletions, insertions or substitutions (e.g., conservativesubstitutions) relative to the amino acid sequence shown in FIG. 30). Inembodiments, the TNFα-binding single domain molecule has one or morebiological activities of the TNFα-binding single domain antibodymolecule shown in FIG. 30. For example, the TNFα-binding single domainmolecule binds to the same or a similar epitope as the epitoperecognized by the TNFα-binding single domain molecule shown in FIG. 30(e.g., binds to TNFα in its trimeric form; binds to the TNFα sitecontacting the TNF receptor; binds to an epitope in the TNFα trimercomprising Gln at position 88 and Lys at position 90 on the first TNFmonomer (monomer A), and Glu at position 146 on the second TNF monomer(monomer B), or an epitope as disclosed in WO 06/122786). In otherembodiment, the TNFα-binding single domain molecule has an activity(e.g., binding affinity, dissociation constant, binding specificity,TNF-inhibitory activity) similar to any of the TNFα-binding singledomain molecule disclosed in WO 06/122786.

In other embodiments, the TNFα-binding nanobody molecule comprises oneor more of the nanobodies disclosed in WO 2006/122786. For example, theTNFα-binding nanobody molecule can be a monovalent, bivalent, trivalentTNFα-binding nanobody molecule disclosed in WO 2006/122786. ExemplaryTNFα-binding nanobodies include, but are not limited to, TNF1, TNF2,TNF3, humanized forms thereof (e.g., TNF29, TNF30, TNF31, TNF32, TNF33).Additional examples of monovalent TNFα-binding nanobodies are disclosedin Table 8 of WO 2006/122786. Exemplary bivalent TNFα-binding nanobodymolecules include, but are not limited to, TNF55 and TNF56, whichcomprise two TNF30 nanobodies linked via a peptide linker to form asingle fusion polypeptide (disclosed in WO 2006/122786). Additionalexamples of bivalent TNFα-binding nanobody molecules are disclosed inTable 19 of WO 2006/122786 as TNF4, TNF5, TNF6, TNF7, TNF8).

In other embodiments, at least one of the single domain molecule of theSDAB molecule of the formulation binds to HSA includes one, two, orthree CDRs having the amino sequence: SFGMS (SEQ ID NO:5) (CDR1),SISGSGSDTLYADSVKG (SEQ ID NO:6) (CDR2) and/or GGSLSR (SEQ ID NO:7)(CDR3), or having a CDR that differs by fewer than 3, 2 or 1 amino acidsubstitutions (e.g., conservative substitutions) from one of said CDRs.In other embodiments, the single domain molecule comprises a variableregion having the amino acid sequence from about amino acids 125 to 239of FIG. 30 (SEQ ID NO:1), or an amino acid sequence substantiallyidentical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%or more identical to, or having up to 20, 15, 10, 5, 4, 3, 2, 1 aminoacid changes (e.g., deletions, insertions or substitutions (e.g.,conservative substitutions) relative to the amino acid sequence shown inFIG. 30 (SEQ ID NO:1)). In embodiments, the HSA-binding single domainmolecule has one or more biological activities of the HSA-binding singledomain molecule shown in FIG. 30 (SEQ ID NO:1). For example, theHSA-binding single domain molecule binds to the same or a similarepitope as the epitope recognized by the HSA-binding single domainmolecule shown in FIG. 30 (SEQ ID NO:1). In other embodiment, theHSA-binding single domain molecule has an activity (e.g., bindingaffinity, dissociation constant, binding specificity) similar to any ofthe HSA-binding single domain molecule disclosed in WO 06/122786.

In other embodiments, the HSA-binding SDAB molecule comprises one ormore of the nanobodies disclosed in WO 2006/122786. For example, theHSA-binding SDAB molecule can be a monovalent, bivalent, trivalentHSA-binding nanobody molecule disclosed in WO 2006/122786. In otherembodiments, the HSA-binding SDAB molecule can be a monospecific or amultispecific molecule having at least one of the binding specificitiesbind to HSA. Exemplary TNFα-binding nanobodies include, but are notlimited to, ALB1, humanized forms thereof (e.g., ALB6, ALB7, ALB8, ALB9,ALB10), disclosed in WO 06/122786.

In other embodiments, two or more of the single domain molecules of theSDAB molecules are fused, with or without a linking group, as a geneticor a polypeptide fusion. The linking group can be any linking groupapparent to those of skill in the art. For instance, the linking groupcan be a biocompatible polymer with a length of 1 to 100 atoms. In oneembodiment, the linking group includes or consists of polyglycine,polyserine, polylysine, polyglutamate, polyisoleucine, or polyarginineresidues, or a combination thereof. For example, the polyglycine orpolyserine linkers can include at least five, seven eight, nine, ten,twelve, fifteen, twenty, thirty, thirty-five and forty glycine andserine residues. Exemplary linkers that can be used include Gly-Serrepeats, for example, (Gly)₄-Ser (SEQ ID NO: 8) repeats of at one, two,three, four, five, six, seven or more repeats. In embodiments, thelinker has the following sequences: (Gly)₄-Ser-(Gly)₃-Ser (SEQ ID NO: 9)or ((Gly)₄-Ser)_(n) (SEQ ID NO: 10), where n is 4, 5, or 6.

The formulations of the invention can include a SDAB molecule that ismodified by associating, e.g., covalently or non-covalently a secondmoiety. For example, the nanobody molecule can be covalently attached toa suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or a derivative thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Examples of pegylated nanobodymolecules are disclosed as TNF55-PEG40, TNF55-PEG60, TNF56-PEG40 andTNF56-PEG60 in WO 06/122786.

In another embodiment, the formulations of the invention are stable forat least 3, 6, 9, 12 months (e.g., at least 24, 30, 36 months), at atemperature of about 2° C. to about 25° C. (e.g., about 4° C. or 25°C.). In certain embodiments, the integrity of the SDAB molecule ismaintained after storage in the formulation for at least at least 3, 6,9, 12 months (e.g., at least 24, 30, 36 months), at a temperature ofabout 2° C. to about 25° C. (e.g., about 4° C. or 25° C.). For example,the SDAB molecule in the formulation retains at least 50%, 70%, 75%,80%, 85%, 90%, 95%, 98% or up to 100% of a biological activity, e.g.,binding activity, of the SDAB molecule after storage at a temperature ofabout 2° C. to about 25° C. (e.g., about 4° C. or 25° C.). In someembodiments, the formulation includes less than 10%, 9%, 5%, 4%, 3%, 2%,1% or less high molecular weight (HMW) species after storage in theformulation for at least at least 3, 6, 9, 12 months (e.g., at least 24,30, 36 months), at a temperature of about 2° C. to about 25° C. (e.g.,about 4° C. or 25° C.). In other embodiments, the formulation includesless than 10%, 9%, 5%, 4%, 3%, 2%, 1% or less low molecular weight (HMW)species after storage in the formulation for at least at least 3, 6, 9,12 months (e.g., at least 24, 30, 36 months), at a temperature of about2° C. to about 25° C. (e.g., about 4° C. or 25° C.). In yet otherembodiments, the formulation includes less than 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1% or less acidic species after storage in the formulationfor at least at least 3, 6, 9, 12 months (e.g., at least 24, 30, 36months), at a temperature of about 2° C. to about 25° C. (e.g., about 4°C. or 25° C.). In yet other embodiments, the formulation includes lessthan 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less basic species afterstorage in the formulation for at least at least 3, 6, 9, 12 months(e.g., at least 24, 30, 36 months), at a temperature of about 2° C. toabout 25° C. (e.g., about 4° C. or 25° C.). The HMW, LMW, acidic andbasic species can be detected in the formulations using standardtechniques, such as size exclusion-high performance liquidchromatography (SEC-HPLC) and the like as described herein. In someembodiments, upon reconstitution of the lyophilized SDAB formulation,the formulation retains at least 80%, 90%, 95% or higher of the SDABstructure compared to the formulation prior to lyophilization. SDABstructure is determined, for example, by binding assay, bioassay, or theratio of HMW species to LMW species.

The formulations of the invention can also include a second agent, e.g.,a second therapeutically or pharmacologically active agent that isuseful in treating a TNF-α associated disorder, e.g., inflammatory orautoimmune disorders, including, but not limited to, rheumatoidarthritis (RA) (e.g., moderate to severe rheumatoid arthritis),arthritic conditions (e.g., psoriatic arthritis, polyarticular juvenileidiopathic arthritis (JIA), ankylosing spondylitis (AS), psoriasis,ulcerative colitis, Crohn's disease, inflammatory bowel disease, and/ormultiple sclerosis. For example, the second agent may be an anti-TNFantibody or TNF binding fragment thereof, wherein the second TNFantibody binds to a different epitope than the TNF-binding SDAB moleculeof the formulation. Other non-limiting examples of agents that can beco-formulated with the TNF-binding SDAB molecule include, but are notlimited to, a cytokine inhibitor, a growth factor inhibitor, animmunosuppressant, an anti-inflammatory agent, a metabolic inhibitor, anenzyme inhibitor, a cytotoxic agent, and a cytostatic agent. In oneembodiment, the additional agent is a standard treatment for arthritis,including, but not limited to, non-steroidal anti-inflammatory agents(NSAIDs); corticosteroids, including prednisolone, prednisone,cortisone, and triamcinolone; and disease modifying anti-rheumatic drugs(DMARDs), such as methotrexate, hydroxychloroquine (Plaquenil) andsulfasalazine, leflunomide (Arava®), tumor necrosis factor inhibitors,including etanercept (Enbrel®), infliximab (Remicade®) (with or withoutmethotrexate), and adalimumab (Humira®), anti-CD20 antibody (e.g.,Rituxan®), soluble interleukin-1 receptor, such as anakinra (Kineret®),gold, minocycline (Minocin®), penicillamine, and cytotoxic agents,including azathioprine, cyclophosphamide, and cyclosporine. Suchcombination therapies may advantageously utilize lower dosages of theadministered therapeutic agents, thus avoiding possible toxicities orcomplications associated with the various monotherapies.

Alternative combination of excipients and/or second therapeutic agentscan be identified and tested followed the guidance provided herein.

In yet another embodiment, the formulations described herein aresuitable for administration to a subject, e.g., a human subject (e.g., apatient having a TNFα associated disorder). The formulation can beadministered to the subject by injection (e.g., subcutaneous,intravascular, intramuscular or intraperitoneal) or by inhalation.

In another aspect, the invention features a method or process ofpreparing the formulations described herein. The method or processincludes: expressing the SDAB molecule in a cell culture; purifying theSDAB molecule, e.g., by passing the SDAB molecule through at least oneof a chromatography purification step, an ultrafiltration/diafiltrationsteps; adjusting the concentration of the SDAB molecule, e.g., to about10 to 250 mg/mL in a formulation containing a lyoprotectant, asurfactant and a buffer as described herein, e.g., sucrose at aconcentration of about 5% to about 10%, e.g., about 5%, about 10%;polysorbate-80 at a concentration of about 0 to about 0.02%, e.g.,0.01%, 0.02%; (optionally) glycine at a concentration of about 0 toabout 100 mM, e.g., 100 mM; (optionally) methionine at a concentrationof about 0 to about 100 mM, e.g., 100 mM; and (f) a Histidine (at aconcentration about 10 to about 20 mM) or a Tris buffer (at aconcentration about 20 mM), such that the pH of the formulation is about5 to 7.5, e.g., 5, 5.5, 5.8-6.1, 6, 6.1, 6.5 or 7.

In another aspect, the invention features a method or process forpreparing a reconstituted formulation containing an SDAB molecule, e.g.,a TNF-binding SDAB molecule as described herein. The method includes:lyophilizing a mixture of an SDAB molecule, a lyoprotectant, asurfactant and a buffer, thereby forming a lyophilized mixture; andreconstituting the lyophilized mixture in a diluent, thereby preparing aformulation as described herein. In one embodiment, the formulationincludes (a) a SDAB molecule, e.g., a TNF-binding nanobody molecule at aconcentration of about 0.5 to about 200 mg/mL, e.g., at about 1 mg/mL,about 50 mg/mL, about 80 mg/mL, about 88 mg/mL, about 100 mg/mL, about118 mg/mL; (b) sucrose at a concentration of about 5% to about 10%,e.g., about 5%, about 10%; (c) polysorbate-80 at a concentration ofabout 0 to about 0.02%, e.g., 0.01%, 0.02%; (d) (optionally) glycine ata concentration of about 0 to about 100 mM, e.g., 100 mM; (e)(optionally) methionine at a concentration of about 0 to about 100 mM,e.g., 100 mM; and (f) a Histidine (at a concentration about 10 to about20 mM) or a Tris buffer (at a concentration about 20 mM), such that thepH of the formulation is about 5 to 7.5, e.g., 5, 5.5, 5.8-6.1, 6, 6.1,6.5 or 7.

In another aspect, the invention relates to a method for treating orpreventing in a subject (e.g., a human subject) a TNFα associateddisorder, e.g., inflammatory or autoimmune disorders, including, but notlimited to, rheumatoid arthritis (RA) (e.g., moderate to severerheumatoid arthritis), arthritic conditions (e.g., psoriatic arthritis,polyarticular juvenile idiopathic arthritis (JIA), ankylosingspondylitis (AS), psoriasis, ulcerative colitis, Crohn's disease,inflammatory bowel disease, and/or multiple sclerosis. The methodincludes administering to a subject, e.g., a human patient, apharmaceutical composition includes a TNF-binding SDAB formulation asdescribed herein, e.g., a formulation containing a TNF-binding SDABmolecule, alone or in combination with any of the combination therapiesdescribed herein, in an amount such that one or more of the symptoms ofthe TNFα associated disorder are reduced.

In another aspect, the invention features a kit or an article ofmanufacture that includes a device, a syringe or a vial containing theformulations described herein. The kit or article may optionally includeinstructions for use. In certain embodiments, the syringe or a vial iscomposed of glass, plastic, or a polymeric material, such as cyclicolefin polymer or copolymer. In other embodiments, the formulation canbe present in an injectable device (e.g., an injectable syringe, e.g., aprefilled injectable syringe). The syringe may be adapted for individualadministration, e.g., as a single vial system including an autoinjector(e.g., a pen-injector device), and/or instructions for use. In oneembodiment, the injectable device is a prefilled pen or other suitableautoinjectable device, optionally with instruction for use andadministration.

In certain embodiments, the kit or article of manufacture (e.g., theprefilled pen or syringe with a single or multiple dose unit) isprovided to a subject, e.g., a patient or a healthcare provider,prepackaged with instructions for administration (e.g.,self-administration) by injection (e.g., subcutaneous, intravascular,intramuscular or intraperitoneal).

In other embodiments, the invention features, a device for nasal,transdermal, intravenous administration of the formulations describedherein is provided. For example, a transdermal patch for administrationof the formulations described herein is provided. In yet other cases, anintravenous bag for administration of the formulations described hereinis provided. In embodiments, the intravenous bag is provided with normalsaline or 5% dextrose.

In another aspect, the invention features a method of instructing apatient (e.g., a human patient) in need of an SDAB molecule, e.g., aTNFα nanobody molecule, how to administer a formulation describedherein. The method includes: (i) providing the patient with at least oneunit dose of a formulation of the SDAB molecule described herein; and(ii) instructing the patient to self-administer the at least one unitdose, e.g., by injection (e.g., subcutaneous, intravascular,intramuscular or intraperitoneal). In one embodiment, the patient has aTNFα associated disorder, e.g., inflammatory or autoimmune disorders asdescribed herein.

In another aspect, the invention features a method of instructing arecipient on the administration of a formulation of TNFα nanobodymolecule described herein. The method includes instructing the recipient(e.g., an end user, patient, physician, retail or wholesale pharmacy,distributor, or pharmacy department at a hospital, nursing home clinicor HMO) how the formulation should be administered to a patient.

In another aspect, a method of distributing a formulation of an SDABmolecule, e.g., a TNFα nanobody molecule, described herein is provided.The method includes providing a recipient (e.g., an end user, patient,physician, retail or wholesale pharmacy, distributor, or pharmacydepartment at a hospital, nursing home clinic or HMO) with a packagecontaining sufficient unit dosages of the SDAB molecule, e.g., a TNFαnanobody molecule, to treat a patient for at least 6, 12, 24, or 36months.

In another aspect, the invention features a method or process ofevaluating the quality of a package or lot of packages (e.g., todetermine if it has expired) of a formulation described hereincontaining a SDAB molecule, e.g., a TNFα nanobody molecule. The methodincludes evaluating whether the package has expired. The expiration dateis at least 6, 12, 24, 36, or 48 months, e.g., greater than 24 or 36months, from a preselected event, such as manufacturing, assaying, orpackaging. In some embodiments, a decision or step is taken as a resultof the analysis, e.g., the SDAB molecule in the package is used ordiscarded, classified, selected, released or withheld, shipped, moved toa new location, released into commerce, sold, or offered for sale,withdrawn from commerce or no longer offered for sale, depending onwhether the product has expired.

In another aspect, the invention features a method of storing,distributing, or using a formulation of an SDAB molecule, e.g., a TNFnanobody molecule, described herein. The method includes: storing theformulation for period at a given temperature, e.g., less than 25° C.,e.g., below freezing or below 15° C., 10° C., or 4° C. In embodiments,the method further includes providing the formulation to a recipient,e.g., an end-user, e.g., a patient or healthcare provider, for storageunder the similar or different conditions (e.g., a higher temperaturethan the first storage period). The formulation can be a liquid,lyophilized or reconstituted formulation.

In another aspect, the invention features a method of analyzing aproduct or a process, e.g., a manufacturing process. The method includesproviding a formulation of an SDAB molecule, e.g., a TNF nanobodymolecule, as described herein, and assessing a parameter of theformulation, such as color (e.g., colorless to slightly yellow, orcolorless to yellow), clarity (e.g., clear to slightly opalescent orclear to opalescent), or viscosity (e.g., between approximately 1 to 5cP when measured at ambient temperature, such as at 20° C.-30° C., e.g.,25° C.), amount of one or more HMW, LMW, acidic and/or basic species, asdescribed herein. The evaluation can include an assessment of one ormore parameters. Optionally, a determination of whether the parametermeets a preselected criteria is determined, e.g., whether thepreselected criteria is present, or is present in a preselected range,is determined, thereby analyzing the process.

In one embodiment, evaluation of the process includes a measure of thestability of the SDAB molecule formulation. Stability of the antibodyformulation can be measured, for example, by aggregate formation, whichis assayed, e.g., by size exclusion high pressure liquid chromatography(SE-HPLC), by color, clarity, or viscosity as described herein. Aformulation can be determined to be stable, and therefore acceptable forfurther processing or distribution, if the change in an assay parameteris less than about 10%, 5%, 3%, 2%, 1%, 0.5%, 0.05%, or 0.005% or less,over a pre-set period of time, and optionally at a given temperature.

In one embodiment, the method further includes comparing the valuedetermined with a reference value, to thereby analyze the manufacturingprocess.

In one embodiment, the method further includes maintaining themanufacturing process based, at least in part, upon the analysis. In oneembodiment, the method further includes altering the manufacturingprocess based upon the analysis.

In another embodiment the method includes evaluating a process, e.g.,manufacturing process, of a formulation of an SDAB molecule, e.g., a TNFnanobody molecule, made by a selected process, that includes making adetermination about the process based upon a method or analysisdescribed herein. In one embodiment, the method further includesmaintaining or altering the manufacturing process based, at least inpart, upon the method or analysis. Thus, in another embodiment the partymaking the evaluation does not practice the method or analysis describedherein but merely relies on results which are obtained by a method oranalysis described herein.

In another embodiment the method includes comparing two or morepreparations in a method of monitoring or controlling batch-to-batchvariation or to compare a preparation to a reference standard.

In yet another embodiment, the method can further include making adecision, e.g., to classify, select, accept or discard, release orwithhold, process into a drug product, ship, move to a differentlocation, formulate, label, package, release into commerce, sell oroffer for sale the preparation, based, at least in part, upon thedetermination.

In another aspect, the invention features a method of evaluating thequality of a formulation of an SDAB molecule, e.g., a TNF nanobodymolecule, as described herein, e.g., in a quality control or releasespecification analysis. The method includes providing an evaluation ofan SDAB molecule formulation for a parameter, such as color (e.g.,colorless to slightly yellow, or colorless to yellow), clarity (e.g.,clear to slightly opalescent or clear to opalescent), or viscosity(e.g., between approximately 1 to 5 cP when measured at ambienttemperature, such as at 20° C. to 30° C., e.g., 25° C.). The evaluationcan include an assessment of one or more of the above parameters. Themethod also includes, optionally, determining whether the solutionparameter meets a preselected criteria, e.g., whether the preselectedcriteria is present, or is present in a preselected range. If theobserved solution parameter is within a preselected range of values, ormeets the preselected standard criteria, then the preparation isselected, such as for packaging, use, sale, release into commerce,discarding etc.

In another aspect, the invention features a method of complying with aregulatory requirement, e.g., a post approval requirement of aregulatory agency, e.g., the FDA. The method includes providing anevaluation of an antibody formulation for a parameter, as describedherein. The post approval requirement can include a measure of one moreof the above parameters. The method also includes, optionally,determining whether the observed solution parameter meets a preselectedcriteria or if the parameter is in a preselected range; optionally,memorializing the value or result of the analysis, or communicating withthe agency, e.g., by transmitting the value or result to the regulatoryagency.

In another aspect, the invention features a method of making a batch ofa formulation of an SDAB molecule, e.g., a TNF nanobody molecule, havinga preselected property, e.g., meeting a release specification, labelrequirement, or compendial requirement, e.g., a property describedherein. The method includes providing a test formulation; analyzing thetest formulation according to a method described herein; determining ifthe test formulation satisfies a preselected criteria, e.g., having apreselected relationship with a reference value, e.g., one or morereference values disclosed herein, and selecting the test antibodypreparation to make a batch of product.

In another aspect, the invention features multiple batches of aformulation of an SDAB molecule, e.g., a TNF nanobody molecule, whereinone or more parameters (e.g., a value or solution parameter determinedby a method described herein), for each batch varies less than apreselected range from a pre-selected desired reference value orcriteria, e.g., a range or criteria described herein. In someembodiments, one or more parameters for one or more batches offormulation, is determined and a batch or batches selected as a resultof the determination. Some embodiments include comparing the results ofthe determination to a preselected value or criteria, e.g., a referencestandard. Other embodiments include adjusting the dose of the batch tobe administered, e.g., based on the result of the determination of thevalue or parameter.

In another aspect, the invention features a method of one or more of:providing a report to a report-receiving entity, evaluating a sample ofa formulation of an SDAB molecule, e.g., a TNF nanobody molecule, forcompliance with a reference standard, e.g., an FDA requirement, seekingindication from another party that a preparation of the SDAB moleculemeets some predefined requirement, or submitting information about apreparation of an SDAB molecule to another party. Exemplary receivingentities or other parties include a government, e.g., the U.S. federalgovernment, e.g., a government agency, e.g., the FDA. The methodincludes one or more (or all) of the following steps for making and/ortesting an aqueous formulation of SDAB molecule in a first country,e.g., the U.S.; sending at least an aliquot of the sample outside thefirst country, e.g., sending it outside the United States, to a secondcountry; preparing, or receiving, a report which includes data about thestructure of the preparation of the SDAB molecule, e.g., data related toa structure and/or chain described herein, e.g., data generated by oneor more of the methods described herein; and providing said report to areport recipient entity.

In one embodiment, the report-receiving entity can determine if apredetermined requirement or reference value is met by the data and,optionally, a response from the report-receiving entity is received,e.g., by a manufacturer, distributor or seller of a formulation of anSDAB molecule. In one embodiment, upon receipt of approval from thereport recipient entity, the preparation of a formulation of an SDABmolecule is selected, packaged, or placed into commerce.

In another aspect, the invention features a method of evaluating aformulation of an SDAB molecule. The method includes receiving data withregard to the presence or level of an SDAB molecule, e.g., wherein thedata was prepared by one or more methods described herein; providing arecord which includes said data and optionally includes an identifierfor a batch of SDAB molecule; submitting said record to adecision-maker, e.g., a government agency, e.g., the FDA; optionally,receiving a communication from said decision maker; optionally, decidingwhether to release or market the batch of SDAB molecule based on thecommunication from the decision maker. In one embodiment, the methodfurther includes releasing the sample.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thedetailed description, drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the results of the biological activity of a lyophilizedformulation of 10⁶ U/mg of TNF-binding nanobody (ATN-103) stored as adried powder (DP) preparation for up to six months. The formulation wasstored at the indicated temperatures.

FIG. 2 depicts the results of Human Serum Albumin (HSA) binding activityof a lyophilized formulation of TNF-binding nanobody (ATN-103). Theresults are shown as percentage (%) of TNF-binding nanobody referencestandard.

FIG. 3 depicts the results for size exclusion-HPLC (SE-HPLC) in terms of% of high molecular weight (HMW) species for lyophilized formulation.

FIG. 4 depicts the results for SDS-capillary electrophoresis (SDS-CE) interms of % TNF-binding nanobody for lyophilized formulation.

FIG. 5 depicts SE-HPLC results for % HMW species for formulationsubjected to control and robustness lyophilization cycles.

FIG. 6 depicts the results of the biological activity of 10⁶ U/mgTNF-binding nanobody after storage for up to six months at a highconcentration liquid formulation.

FIG. 7 depicts results of Human Serum Albumin (HSA) binding activity(percentage of TNF-binding nanobody Reference Standard) of highconcentration liquid formulation stored for up to six months at thetemperatures indicated.

FIG. 8 depicts SE-HPLC results for % HMW species of high concentrationliquid formulation after storage for up to six months at thetemperatures indicated.

FIG. 9 depicts SE-HPLC results for % LMW species of high concentrationliquid formulation after storage for up to six months at thetemperatures indicated.

FIG. 10 depicts SDS-CE results for % ATN-103 of high concentrationliquid formulation after storage for up to six months at thetemperatures indicated.

FIG. 11 depicts SE-HPLC results for % HMW species of high concentrationliquid formulation in a prefilled syringe.

FIG. 12 depicts SE-HPLC results for % LMW species of high concentrationliquid formulation in a prefilled syringe.

FIG. 13 depicts results for % acidic species by CEX-HPLC of highconcentration liquid formulation in a prefilled syringe.

FIG. 14 depicts results for % basic species by CEX-HPLC of highconcentration liquid formulation in a prefilled syringe.

FIG. 15 depicts SE-HPLC results for % HMW species of high concentrationliquid formulations—Other Formulations (identification of otherstabilizing and destabilizing excipients).

FIG. 16 depicts SE-HPLC results for % HMW species for TNF-bindingnanobody high concentration liquid.

FIG. 17 depicts SE-HPLC results for % LMW species for TNF-bindingnanobody high concentration liquid.

FIG. 18 depicts CEX-HPLC results for % Acidic species for TNF-bindingnanobody high concentration liquid.

FIG. 19 depicts CEX-HPLC results for % Basic species for TNF-bindingnanobody high concentration liquid.

FIG. 20 depicts SE-HPLC results for % HMW species for TNF-bindingnanobody high concentration liquid after 10× freeze-thaw cycles.

FIG. 21 depicts SE-HPLC results for % LMW species for TNF-bindingnanobody high concentration liquid after 10× freeze-thaw cycles.

FIG. 22 depicts Turbidity (Absorbance at 455 nm) results for TNF-bindingnanobody high concentration liquid after 10× freeze-thaw cycles.

FIG. 23 depicts Concentration (by UV absorbance at 280 nm) results forTNF-binding nanobody high concentration liquid after 10× freeze-thawcycles.

FIG. 24 depicts High Concentration Liquid Formulation of TNF-bindingnanobody: % HMW by SE-HPLC after of short term thermal stressespotentially encountered in manufacturing processes.

FIG. 25 depicts SE-HPLC results for % HMW species of low concentrationliquid formulation as a function of pH and formulation (40° C.).

FIG. 26 depicts SE-HPLC results for % LMW species of low concentrationliquid formulation as a function of pH and formulation (40° C.).

FIG. 27 depicts SE-HPLC results for % HMW species of low concentrationliquid formulation as a function of pH and formulation (4° C.).

FIG. 28 depicts SE-HPLC results for % HMW species of low concentrationliquid formulation as a function of pH and formulation after shaking.

FIG. 29 depicts a schematic diagram of the predicted structure ofATN-103.

FIG. 30 depicts the amino acid sequence of ATN-103 polypeptide chain(SEQ ID NO:1).

FIG. 31 are bar graphs depicting the % of HMW species detected bySE-HPLC of the indicated formulations containing approximately 100 mg/mlof ATN-103 (HST, HSGT, HSGMT, HSorb and control) stored under theconditions indicated.

FIG. 32 are bar graphs depicting the % of LMW species detected bySE-HPLC of the indicated formulations containing approximately 100 mg/mlof ATN-103 (HST, HSGT, HSGMT, HSorb and control) stored under theconditions indicated. No LMW species was detected at the initial timepoint or after two weeks at 4° C.

DETAILED DESCRIPTION

Stable formulations that include an SDAB molecule, e.g., a nanobodymolecule (e.g., a TNF-binding nanobody molecule), have been identifiedthat are suitable for storage of high and low concentrations of the SDABmolecule (a “formulation”). The SDAB molecule which is formulated ispreferably essentially pure and desirably essentially homogeneous (i.e.free from contaminating proteins etc). “Essentially pure” protein meansa composition comprising at least about 90% by weight of the protein,based on total weight of the composition, preferably at least about 95%by weight. “Essentially homogeneous” protein means a compositioncomprising at least about 99% by weight of protein, based on totalweight of the composition.

The integrity of the SDAB molecule in the formulation is generallymaintained following long-term storage as a liquid or as a lyophilizedproduct under various conditions. For example, the integrity of the SDABmolecule is adequately maintained after exposure to a wide range ofstorage temperatures (e.g., −80° C. to 40° C.), shear stress (e.g.,shaking) and interfacial stress (freeze-thaw cycles).

Additionally, for lyophilized material, the integrity of the SDABmolecule is adequately maintained during the process of reconstitution.In addition, SDAB molecule integrity is sufficiently maintained for useas a medicament as demonstrated by relatively low accumulations of LMWspecies and HMW species, bioactivity in vitro, binding activity invitro, after long term storage (e.g., up to 12 months) at varioustemperatures (e.g., −80° C. to 40° C.).

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, the articles “a” and “an” refer to one or to more thanone (e.g., to at least one) of the grammatical object of the article.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or”, unless context clearly indicates otherwise.

The terms “proteins” and “polypeptides” are used interchangeably herein.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values.

A “stable” formulation of an SDAB molecule exhibits little or no signsof any one or more of aggregation, fragmentation, deamidation,oxidation, or change in biological activity over an extended period oftime, e.g., 6, 12 months, 24 months, 36 months or longer. For example,in one embodiment, less than 10% of the SDAB molecule is aggregated,fragmented, or oxidated. Aggregation, precipitation, and/or denaturationcan be assessed by known methods, such as visual examination of colorand/or clarity, or by UV light scattering or size exclusionchromatography. The ability of the protein to retain its biologicalactivity can be assessed by detecting and quantifying chemically alteredforms of the antibody. Size modification (e.g., clipping), which can beevaluated using size exclusion chromatography, and/or SDS-PAGE, forexample. Other types of chemical alteration include charge alteration(e.g., occurring as a result of deamidation), which can be evaluated byion-exchange chromatography, for example.

An SDAB molecule “retains its biological activity” in a pharmaceuticalformulation, if the biological activity of the molecule at a given timeis within about 50% or higher of the biological activity exhibited atthe time the pharmaceutical formulation was prepared as determined in anantigen binding assay, for example.

A “reconstituted” formulation is one which has been prepared bydissolving a lyophilized protein formulation in a diluent such that theprotein is dispersed in the reconstituted formulation. The reconstitutedformulation in suitable for administration (e.g. parenteral orperipheral administration) to a patient to be treated with the proteinof interest and, in certain embodiments of the invention, may be onewhich is suitable for subcutaneous administration.

By “isotonic” or “iso-osmotic” is meant that the formulation of interesthas similar or essentially the same osmotic pressure as human blood.Isotonic or iso-osmotic formulations will generally have an osmoticpressure from about 250 to 350 mOsm. Isotonicity can be measured using avapor pressure or ice-freezing type osmometer, for example.

A “tonicity adjusting agent” refers to a compound that renders theformulation substantially isotonic or iso-osmotic with human blood.Exemplary tonicity adjusting agents are: sucrose, sorbitol, glycine,methionine, mannitol, dextrose, inositol, sodium chloride, arginine, orarginine hydrochloride. Typically, tonicity adjusting agents are addedin an amount such that the overall formulation exerts an osmoticstrength similar to that of human blood. For example, human bloodcontains approximately 300 mM solutes. Typically, pharmaceuticalproducts target a total molarity of 300 mM. This corresponds to anosmotic pressure of approximately 300 to 310 mOsm, with a typical rangeof 250 mOsm to 350 mOsm. The amount of tonicity adjusting agent requiredcan be initially estimated via calculation. The contribution to totalmolarity can be estimated from molecular weight of the excipientmolecule, and known properties of the molecule, e.g. does the moleculedissociate into two ionic species, or is the molecule non-ionic (doesnot dissociate). Additionally, it is necessary to understand the osmoticcontribution of the specific protein molecule as a function of proteinconcentration. This parameter can be determined experimentally.

For example, starting with a formulation (not tonicity corrected) of 10mM histidine, 5% sucrose, 0.01% polysorbate 80, with an anti-TNFnanobody protein concentration of 100 mg/mL, as a first step, theestimated molarity of the starting formulation can be calculated asfollows:

10 mM histidine=10 mM

5% sucrose corresponds to approximately 146 mM

5%=5 g/100 mL=50 g/L→(50 g/L)/(342.3 g/mol)=0.146 mol/L=146 mM 0.01%polysorbate 80 exerts essentially zero molarity and can be disregarded.100 mg/mL protein: It has been determined through experimentation that100 mg/mL anti-TNF nanobody protein exerts an osmotic pressure thatcorresponds to approximately 48 mM.

Therefore, summing all contributions to molarity in the initialformulation:

10 mM+146 mM+48 mM=204 mM

If the target molarity is 310 mM, then the corresponding amount molarityto make up the remainder of the target is:

310 mM−204 mM=106 mM

Thus, the recommended amount of tonicity adjusting agent is 106 mM of anon-ionic tonicity adjusting agent, or 53 mM of an ionic tonicityadjusting agent that completely dissociates into two ionic species.

After the initial estimate of tonicity adjusting agent is determined, itis recommended to test the formulation experimentally. Thus, in theexample provided, 100 mM glycine was added to the initial formulation.(The recommended 106 mM was rounded down to 100 mM for simplicity). Theexpected osmolarity would be: 10 mM histidine+146 mM sucrose+48 mMprotein+100 mM glycine=304 mM The experimental osmotic pressure value ofthe formulation=305 mOsm.

A “lyoprotectant” is a molecule which, when combined with a protein ofinterest, significantly prevents or reduces chemical and/or physicalinstability of the protein upon lyophilization and subsequent storage.Exemplary lyoprotectants include sugars such as sucrose, sorbitol, ortrehalose; an amino acid such as monosodium glutamate or histidine; amethylamine such as betaine; a lyotropic salt such as magnesium sulfate;a polyol such as trihydric or higher sugar alcohols, e.g. glycerin,erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol;propylene glycol; polyethylene glycol; Pluronics; and combinationsthereof. Typically, the lyoprotectant is a non-reducing sugar, such astrehalose or sucrose. The lyoprotectant is added to the pre-lyophilizedformulation in a “lyoprotecting amount” which means that, followinglyophilization of the protein in the presence of the lyoprotectingamount of the lyoprotectant, the protein essentially retains itsphysical and chemical stability and integrity upon lyophilization andstorage.

A “stabilizer” refers to a molecule which, when combined with a proteinof interest (e.g., the SDAB molecule) substantially prevents or reduceschemical and/or physical instability of the protein of interest inlyophilized, reconstituted, liquid or storage form. Exemplarystabilizers include sucrose, sorbitol, glycine, inositol, sodiumchloride, methionine, arginine, and arginine hydrochloride.

The “diluent” of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a reconstituted formulation. Exemplarydiluents include sterile water, bacteriostatic water for injection(BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterilesaline solution, Ringer's solution or dextrose solution.

A “preservative” is a compound which can be added to the diluent toessentially reduce bacterial action in the reconstituted formulation,thus facilitating the production of a multi-use reconstitutedformulation, for example. Examples of potential preservatives includeoctadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,benzalkonium chloride (a mixture of alkylbenzyldimethylammoniumchlorides in which the alkyl groups are long-chain compounds), andbenzethonium chloride. Other types of preservatives include aromaticalcohols such as phenol, butyl and benzyl alcohol, alkyl parabens suchas methyl or propyl paraben, catechol, resorcinol, cyclohexanol,3-pentanol, and m-cresol. The most preferred preservative herein isbenzyl alcohol.

A “bulking agent” is a compound which adds mass to the lyophilizedmixture and contributes to the physical structure of the lyophilizedcake (e.g. facilitates the production of an essentially uniformlyophilized cake which maintains an open pore structure). Exemplarybulking agents include mannitol, glycine, polyethylene glycol andxorbitol.

The methods and compositions of the present invention encompasspolypeptides and nucleic acids having the sequences specified, orsequences substantially identical or similar thereto, e.g., sequences atleast 85%, 90%, 95% identical or higher to the sequence specified. Inthe context of an amino acid sequence, the term “substantiallyidentical” is used herein to refer to a first amino acid that contains asufficient or minimum number of amino acid residues that are i)identical to, or ii) conservative substitutions of aligned amino acidresidues in a second amino acid sequence such that the first and secondamino acid sequences can have a common structural domain and/or commonfunctional activity. For example, amino acid sequences containing acommon structural domain having at least about 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence. Inother embodiments, the amino acid sequence can contain one or more aminoacid insertions, deletions, or substitutions (e.g., conservativesubstitutions) to arrive at a percentage identity of at least about 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to areference sequence

In the context of nucleotide sequence, the term “substantiallyidentical” is used herein to refer to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Forexample, nucleotide sequences having at least about 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative” and“analog” when referring to proteins of the present invention include anypolypeptides which retain at least some of the functional properties ofthe corresponding native antibody or polypeptide. Fragments ofpolypeptides of the present invention include proteolytic fragments, aswell as deletion fragments, in addition to specific antibody fragmentsdiscussed elsewhere herein. Variants of the polypeptides of the presentinvention include fragments as described above, and also polypeptideswith altered amino acid sequences due to amino acid substitutions,deletions, or insertions. Variants may occur naturally or benon-naturally occurring. Non-naturally occurring variants may beproduced using art-known mutagenesis techniques. Variant polypeptidesmay comprise conservative or non-conservative amino acid substitutions,deletions or additions. Derivatives of the fragments of the presentinvention are polypeptides which have been altered so as to exhibitadditional features not found on the native polypeptide. Examplesinclude fusion proteins. Variant polypeptides may also be referred toherein as “polypeptide analogs.” As used herein a “derivative” of apolypeptide refers to a subject polypeptide having one or more residueschemically derivatized by reaction of a functional side group. Alsoincluded as “derivatives” are those polypeptides which contain one ormore naturally occurring amino acid derivatives of the twenty standardamino acids. For example, 4-hydroxyproline may be substituted forproline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.

The term “functional variant” refers polypeptides that have asubstantially identical amino acid sequence to the naturally-occurringsequence, or are encoded by a substantially identical nucleotidesequence, and are capable of having one or more activities of thenaturally-occurring sequence.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, 60%, and even more preferably at least 70%,80%, 90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”).

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused unless otherwise specified) are a Blossum 62 scoring matrix with agap penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller ((1989)CABIOS, 4:11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid (SEQ ID NO:1) molecules of the invention. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to a protein (SEQ ID NO:1)protein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizingBLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

Various aspects of the invention are described in further detail below.

Single Domain Antigen Binding (SDAB) Molecules

Single domain antigen binding (SDAB) molecules include molecules whosecomplementary determining regions are part of a single domainpolypeptide. Examples include, but are not limited to, heavy chainvariable domains, binding molecules naturally devoid of light chains,single domains derived from conventional 4-chain antibodies, engineereddomains and single domain scaffolds other than those derived fromantibodies. SDAB molecules may be any of the art, or any future singledomain molecules. SDAB molecules may be derived from any speciesincluding, but not limited to mouse, human, camel, llama, fish, shark,goat, rabbit, and bovine. This term also includes naturally occurringsingle domain antibody molecules from species other than Camelidae andsharks.

In one aspect of the invention, an SDAB molecule can be derived from avariable region of the immunoglobulin found in fish, such as, forexample, that which is derived from the immunoglobulin isotype known asNovel Antigen Receptor (NAR) found in the serum of shark. Methods ofproducing single domain molecules derived from a variable region of NAR(“IgNARs”) are described in WO 03/014161 and Streltsov (2005) ProteinSci. 14:2901-2909.

According to another aspect of the invention, an SDAB molecule is anaturally occurring single domain antigen binding molecule known asheavy chain devoid of light chains. Such single domain molecules aredisclosed in WO 9404678 and Hamers-Casterman, C. et al. (1993) Nature363:446-448, for example. For clarity reasons, this variable domainderived from a heavy chain molecule naturally devoid of light chain isknown herein as a VHH or nanobody to distinguish it from theconventional VH of four chain immunoglobulins. Such a VHH molecule canbe derived from Camelidae species, for example in camel, llama,dromedary, alpaca and guanaco. Other species besides Camelidae mayproduce heavy chain molecules naturally devoid of light chain; such VHHsare within the scope of the invention.

The SDAB molecules can be recombinant, CDR-grafted, humanized,camelized, de-immunized and/or in vitro generated (e.g., selected byphage display), as described in more detail below.

The term “antigen-binding” is intended to include the part of apolypeptide, e.g., a single domain molecule described herein, thatcomprises determinants that form an interface that binds to a targetantigen, or an epitope thereof. With respect to proteins (or proteinmimetics), the antigen-binding site typically includes one or more loops(of at least four amino acids or amino acid mimics) that form aninterface that binds to the target antigen. Typically, theantigen-binding site of the polypeptide, e.g., the single domainantibody molecule, includes at least one or two CDRs, or more typicallyat least three, four, five or six CDRs.

The term “immunoglobulin variable domain” is frequently understood inthe art as being identical or substantially identical to a VL or a VHdomain of human or animal origin. It shall be recognized thatimmunoglobulin variable domain may have evolved in certain species,e.g., sharks and llama, to differ in amino acid sequence from human ormammalian VL or VH. However, these domains are primarily involved inantigen binding. The term “immunoglobulin variable domain” typicallyincludes at least one or two CDRs, or more typically at least threeCDRs.

A “constant immunoglobulin domain” or “constant region” is intended toinclude an immunoglobulin domain that is identical to or substantiallysimilar to a CL, CH1, CH2, CH3, or CH4, domain of human or animalorigin. See e.g. Charles A Hasemann and J. Donald Capra,Immunoglobulins: Structure and Function, in William E. Paul, ed.,Fundamental Immunology, Second Edition, 209, 210-218 (1989). The term“Fc region” refers to the Fc portion of the constant immunoglobulindomain that includes immunoglobulin domains CH2 and CH3 orimmunoglobulin domains substantially similar to these.

In certain embodiments, the SDAB molecule is a monovalent, or amultispecific molecule (e.g., a bivalent, trivalent, or tetravalentmolecule). In other embodiments, the SDAB molecule is a monospecific,bispecific, trispecific or tetraspecific molecule. Whether a molecule is“monospecific” or “multispecific,” e.g., “bispecific,” refers to thenumber of different epitopes with which a binding polypeptide reacts.Multispecific molecules may be specific for different epitopes of atarget polypeptide described herein or may be specific for a targetpolypeptide as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material.

As used herein the term “valency” refers to the number of potentialbinding domains, e.g., antigen binding domains, present in an SDABmolecule. Each binding domain specifically binds one epitope. When anSDAB molecule comprises more than one binding domain, each bindingdomain may specifically bind the same epitope, for an antibody with twobinding domains, termed “bivalent monospecific,” or to differentepitopes, for an SDAB molecule with two binding domains, termed“bivalent bispecific.” An SDAB molecule may also be bispecific andbivalent for each specificity (termed “bispecific tetravalentmolecules”). Bispecific bivalent molecules, and methods of making them,are described, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706;5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537, thedisclosures of all of which are incorporated by reference herein.Bispecific tetravalent molecules, and methods of making them aredescribed, for instance, in WO 02/096948 and WO 00/44788, thedisclosures of both of which are incorporated by reference herein. Seegenerally, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO92/05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos.4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al.,J. Immunol. 148:1547-1553 (1992).

In certain embodiments, the SDAB molecule is a single chain fusionpolypeptide comprising one or more single domain molecules (e.g.,nanobodies), devoid of a complementary variable domain or animmunoglobulin constant, e.g., Fc, region, that binds to one or moretarget antigens. An exemplary target antigen recognized by theantigen-binding polypeptides includes tumor necrosis factor cc (TNF cc).In certain embodiments, the antigen-binding single domain molecule bindsto a serum protein, e.g., a human serum proteins chosen from one or moreof serum albumin (human serum albumin (HSA)) or transferin.

TNFα

Tumor necrosis factor alpha is known in the art to the associated withinflammatory disorders such as rheumatoid arthritis, Crohn's disease,ulcerative colitis and multiple sclerosis. Both TNFα and the receptors(CD120a and CD120b) have been studied in great detail. TNFα in itsbioactive form is a trimer. Several strategies to antagonize the actionof TNFα using anti-TNFα antibodies have been developed and are currentlycommercially available, such as Remicade® and Humira®. Antibodymolecules against TNFα are known. Numerous examples of TNFα-bindingsingle domain antigen binding molecules (e.g., nanobodies) are disclosedin WO 2004/041862,

WO 2004/041865, WO 2006/122786, the contents of all of which areincorporated by reference herein in their entirety. Additional examplesof single domain antigen binding molecules are disclosed in US2006/286066, US 2008/0260757, WO 06/003388, US 05/0271663, US06/0106203, the contents of all of which are incorporated by referenceherein in their entirety. In other embodiments, mono-, bi-, tri- andother multi-specific single domain antibodies against TNFα and a serumprotein, e.g., HSA, are disclosed in these references.

In specific embodiments, the TNFα-binding nanobody molecule comprisesone or more of the nanobodies disclosed in WO 2006/122786. For example,the TNFα-binding nanobody molecule can be a monovalent, bivalent,trivalent TNFα-binding nanobody molecule disclosed in WO 2006/122786.Exemplary TNFα-binding nanobodies include, but are not limited to, TNF1,TNF2, TNF3, humanized forms thereof (e.g., TNF29, TNF30, TNF31, TNF32,TNF33). Additional examples of monovalent TNFα-binding nanobodies aredisclosed in Table 8 of WO 2006/122786. Exemplary bivalent TNFα-bindingnanobody molecules include, but are not limited to, TNF55 and TNF56,which comprise two TNF30 nanobodies linked via a peptide linker to forma single fusion polypeptide (disclosed in WO 2006/122786). Additionalexamples of bivalent TNFα-binding nanobody molecules are disclosed inTable 19 of WO 2006/122786 as TNF4, TNF5, TNF6, TNF7, TNF8).

In other embodiments, the HSA-binding nanobody molecule comprises one ormore of the nanobodies disclosed in WO 2006/122786. For example, theHSA-binding nanobody molecule can be a monovalent, bivalent, trivalentHSA-binding nanobody molecule disclosed in WO 2006/122786. In otherembodiments, the HSA-binding nanobody molecule can be a monospecific ora multispecific molecule having at least one of the bindingspecificities bind to HSA. Exemplary TNFα-binding nanobodies include,but are not limited to, ALB1, humanized forms thereof (e.g., ALB6, ALB7,ALB8, ALB9, ALB10), disclosed in WO 06/122786.

In other embodiments, two or more of the single domain molecules of thenanobody molecules are fused, with or without a linking group, as agenetic or a polypeptide fusion. The linking group can be any linkinggroup apparent to those of skill in the art. For instance, the linkinggroup can be a biocompatible polymer with a length of 1 to 100 atoms. Inone embodiment, the linking group includes or consists of polyglycine,polyserine, polylysine, polyglutamate, polyisoleucine, or polyarginineresidues, or a combination thereof. For example, the polyglycine orpolyserine linkers can include at least five, seven eight, nine, ten,twelve, fifteen, twenty, thirty, thirty-five and forty glycine andserine residues. Exemplary linkers that can be used include Gly-Serrepeats, for example, (Gly)₄-Ser (SEQ ID NO: 8) repeats of at one, two,three, four, five, six, seven or more repeats. In embodiments, thelinker has the following sequences: (Gly)₄-Ser-(Gly)₃-Ser (SEQ ID NO: 9)or ((Gly)₄-Ser)n (SEQ ID NO: 10), where n is 4, 5, or 6.

In one exemplary embodiment, an antigen-binding polypeptide composed ofa single chain polypeptide fusion of two single domain antibodymolecules (e.g., two camelid variable regions) that bind to a targetantigen, e.g., tumor necrosis factor alpha (TNFa), and one single domainantibody molecule (e.g., a camelid variable region) that binds to aserum protein, e.g., HSA, referred to herein as “ATN-103,” was shown tobind to Protein A, or a functional variant thereof. ATN-103 is ahumanized, trivalent, bi-specific, TNFα-inhibiting fusion protein. Theantigen for this protein is tumor necrosis factor-alpha (TNF). FIG. 29provides a schematic representation of the predicted structure ofATN-103. This fusion protein is derived from camelids and has a highdegree of sequence and structural homology to human immunoglobulin VHdomains. Its single polypeptide chain is composed of two binding domainsto TNFα and one to human serum albumin (HSA), with two nine amino acidG-S linkers connecting the domains. A detailed description of ATN-103 isprovided in WO 06/122786.

The complete amino acid sequence of the ATN-103 polypeptide chainpredicted from the DNA sequence of the corresponding expression vectoris shown in FIG. 30 (residues are numbered starting with theNH₂-terminus as Residue Number 1 of SEQ ID NO:1). The last amino acidresidue encoded by the DNA sequence is S³⁶³ and constitutes theCOOH-terminus of the protein. The predicted molecular mass fordisulfide-bonded ATN-103 (with no posttranslational modifications) is38434.7 Da. ATN-103 contains no N-linked glycosylation consensussequence. The molecular mass observed for the predominant isoform bynanoelectrospray ionization quadrupole time-of-flight mass spectrometrycorresponds to 38433.9 Da confirming the absence of post-translationalmodifications.

In FIG. 30, complementarity determining regions (CDR) are underlined.The predicted intramolecular disulfide bonds are illustrated byconnections of the cysteine residues involved. The binding domains toTNF are shown in bold and the binding domain to HSA is shown in bolditalics. The amino acid linkers connecting these binding domains are initalics. The signal peptide (⁻¹⁹MGW . . . VHS⁻¹) is also shown for thepolypeptide chain.

Preparation of SDAB Molecules

The SDAB molecules may comprised of one or more single domain molecules(e.g., nanobodies) that are recombinant, CDR-grafted, humanized,camelized, de-immunized, and/or in vitro generated (e.g., selected byphage display). Techniques for generating antibodies and SDAB molecules,and modifying them recombinantly are known in the art and are describedin detail below.

Numerous methods known to those skilled in the art are available forobtaining antibodies. For example, monoclonal antibodies may be producedby generation of hybridomas in accordance with known methods. Hybridomasformed in this manner are then screened using standard methods, such asenzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance(BIACORE™) analysis, to identify one or more hybridomas that produce annanobody that specifically binds with a specified antigen. Any form ofthe specified antigen may be used as the immunogen, e.g., recombinantantigen, naturally occurring forms, any variants or fragments thereof,as well as antigenic peptide thereof.

One exemplary method of making antibodies and SDAB molecules includesscreening protein expression libraries, e.g., phage or ribosome displaylibraries. Phage display is described, for example, in Ladner et al.,U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO92/01047; WO 92/09690; and WO 90/02809.

In addition to the use of display libraries, the specified antigen canbe used to immunize a non-human animal, e.g., a rodent, e.g., a mouse,hamster, or rat. In one embodiment, the non-human animal includes atleast a part of a human immunoglobulin gene. For example, it is possibleto engineer mouse strains deficient in mouse antibody production withlarge fragments of the human Ig loci. Using the hybridoma technology,antigen-specific monoclonal antibodies derived from the genes with thedesired specificity may be produced and selected. See, e.g., XENOMOUSE™,Green et al. (1994) Nature Genetics 7:13-21, US 2003-0070185, WO96/34096, published Oct. 31, 1996, and PCT Application No.PCT/US96/05928, filed Apr. 29, 1996.

In another embodiment, an SDAB molecule is obtained from the non-humananimal, and then modified, e.g., humanized, deimmunized, chimeric, maybe produced using recombinant DNA techniques known in the art. A varietyof approaches for making chimeric antibodies and SDAB molecules havebeen described. See e.g., Morrison et al., Proc. Natl. Acad. Sci. U.S.A.81:6851, 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., U.S.Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi etal., European Patent Publication EP171496; European Patent Publication0173494, United Kingdom Patent GB 2177096B. Humanized antibodies andSDAB molecules may also be produced, for example, using transgenic micethat express human heavy and light chain genes, but are incapable ofexpressing the endogenous mouse immunoglobulin heavy and light chaingenes. Winter describes an exemplary CDR-grafting method that may beused to prepare the humanized antibodies and SDAB molecule describedherein (U.S. Pat. No. 5,225,539). All of the CDRs of a particular humanantibody may be replaced with at least a portion of a non-human CDR, oronly some of the CDRs may be replaced with non-human CDRs. It is onlynecessary to replace the number of CDRs required for binding of thehumanized antibody and SDAB molecule to a predetermined antigen.

Humanized antibodies can be generated by replacing sequences of the Fvvariable domain that are not directly involved in antigen binding withequivalent sequences from human Fv variable domains. Exemplary methodsfor generating humanized antibodies or fragments thereof are provided byMorrison (1985) Science 229:1202-1207; by Oi et al. (1986) BioTechniques4:214; and by U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S.Pat. No. 5,693,762; U.S. Pat. No. 5,859,205; and U.S. Pat. No.6,407,213. Those methods include isolating, manipulating, and expressingthe nucleic acid sequences that encode all or part of immunoglobulin Fvvariable domains from at least one of a heavy or light chain. Suchnucleic acids may be obtained from a hybridoma producing an nanobodyagainst a predetermined target, as described above, as well as fromother sources. The recombinant DNA encoding the humanized SDAB molecule,e.g., nanobody molecule, can then be cloned into an appropriateexpression vector.

In certain embodiments, a humanized SDAB molecule, e.g., nanobodymolecule, is optimized by the introduction of conservativesubstitutions, consensus sequence substitutions, germline substitutionsand/or backmutations. Such altered immunoglobulin molecules can be madeby any of several techniques known in the art, (e.g., Teng et al., Proc.Natl. Acad. Sci. U.S.A., 80: 7308-7312, 1983; Kozbor et al., ImmunologyToday, 4: 7279, 1983; Olsson et al., Meth. Enzymol., 92: 3-16, 1982),and may be made according to the teachings of PCT Publication WO92/06193or EP 0239400).

Techniques for humanizing SDAB molecules, e.g., nanobody molecules, aredisclosed in WO 06/122786.

An SDAB molecule, e.g., nanobody molecule, may also be modified byspecific deletion of human T cell epitopes or “deimmunization” by themethods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy andlight chain variable domains of, e.g., a nanobody can be analyzed forpeptides that bind to MHC Class II; these peptides represent potentialT-cell epitopes (as defined in WO 98/52976 and WO 00/34317). Fordetection of potential T-cell epitopes, a computer modeling approachtermed “peptide threading” can be applied, and in addition a database ofhuman MHC class II binding peptides can be searched for motifs presentin the V_(H) and V_(L) sequences, as described in WO 98/52976 and WO00/34317. These motifs bind to any of the 18 major MHC class II DRallotypes, and thus constitute potential T cell epitopes. PotentialT-cell epitopes detected can be eliminated by substituting small numbersof amino acid residues in the variable domains, or preferably, by singleamino acid substitutions. Typically, conservative substitutions aremade. Often, but not exclusively, an amino acid common to a position inhuman germline antibody sequences may be used. Human germline sequences,e.g., are disclosed in Tomlinson, et al. (1992) J. Mol. Biol.227:776-798; Cook, G. P. et al. (1995) Immunol. Today Vol. 16 (5):237-242; Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; andTomlinson et al. (1995) EMBO J. 14:4628-4638. The V BASE directoryprovides a comprehensive directory of human immunoglobulin variableregion sequences (compiled by Tomlinson, I. A. et al. MRC Centre forProtein Engineering, Cambridge, UK). These sequences can be used as asource of human sequence, e.g., for framework regions and CDRs.Consensus human framework regions can also be used, e.g., as describedin U.S. Pat. No. 6,300,064.

The SDAB molecules, e.g., nanobody molecules, can be produced by livinghost cells that have been genetically engineered to produce the protein.Methods of genetically engineering cells to produce proteins are wellknown in the art. See e.g. Ausabel et al., eds. (1990), CurrentProtocols in Molecular Biology (Wiley, New York). Such methods includeintroducing nucleic acids that encode and allow expression of theprotein into living host cells. These host cells can be bacterial cells,fungal cells, or, preferably, animal cells grown in culture. Bacterialhost cells include, but are not limited to, Escherichia coli cells.Examples of suitable E. coli strains include: HB101, DH5a, GM2929,JM109, KW251, NM538, NM539, and any E. coli strain that fails to cleaveforeign DNA. Fungal host cells that can be used include, but are notlimited to, Saccharomyces cerevisiae, Pichia pastoris and Aspergilluscells. A few examples of animal cell lines that can be used are CHO,VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, and W138. New animal cell linescan be established using methods well know by those skilled in the art(e.g., by transformation, viral infection, and/or selection).Optionally, the protein can be secreted by the host cells into themedium.

Modified SDAB Molecules

The formulations of the invention may contain at least one SDABmolecule, e.g., nanobody molecule, having an amino acid sequence thatdiffers at least one amino acid position in one of the framework regionsfrom the amino acid sequence of a naturally occurring domain, e.g., VHdomain.

It shall be understood that the amino acid sequences of the some of theSDAB molecules of the invention, such as the humanized SDAB molecules,can differ at least one amino acid position in at least one of theframework regions from the amino acid sequences of naturally occurringdomain, e.g., a naturally occurring VHI-I domains.

The invention also includes formulations of derivatives of the SDABmolecules. Such derivatives can generally be obtained by modification,and in particular by chemical and/or biological (e.g enzymatical)modification, of the SDAB molecules and/or of one or more of the aminoacid residues that form the SDAB molecules disclosed herein.

Examples of such modifications, as well as examples of amino acidresidues within the SDAB molecule sequence that can be modified in sucha manner (i.e. either on the protein backbone but preferably on a sidechain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the SDAB molecule,and in particular of one or more functional groups, residues or moietiesthat confer one or more desired properties or functionalities to theSDAB molecules. Example of such functional groups will be clear to theskilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that that increase the half-life, the solubilityand/or the absorption of the SDAB molecule, that reduce theimmunogenicity and/or the toxicity of the SDAB molecule, that eliminateor attenuate any undesirable side effects of the SDAB molecule, and/orthat confer other advantageous properties to and/or reduce the undesiredproperties of the SDAB molecule; or any combination of two or more ofthe foregoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and-148-single domain antibodies), for whichreference is for example made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may for example be linked directly (for example covalently) to aNanobody of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One widely used techniques for increasing the half-life and/or thereducing immunogenicity of pharmaceutical proteins comprises attachmentof a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman,Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug.Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylationof proteins are also commercially available, for example from NektarTherapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering, 16,10, 761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in an SDAB molecule, an SDABmolecule may be modified so as to suitably introduce one or morecysteine residues for attachment of PEG, or an amino acid sequencecomprising one or more cysteine residues for attachment of PEG may befused to the N- and/or C-terminus of a Nanobody of the invention, allusing techniques of protein engineering known per se to the skilledperson.

Preferably, for the SDAB molecule, a PEG is used with a molecular weightof more than 5000, such as more than 10,000 and less than 200,000, suchas less than 100,000; for example in the range of 20,000-80,000.

With regard to pegylation, its should be noted that generally, theinvention also encompasses any SDAB molecule that has been pegylated atone or more amino acid positions, preferably in such a way that saidpegylation either (1) increases the half-life in vivo; (2) reducesimmunogenicity; (3) provides one or more further beneficial propertiesknown per se for pegylation; (4) does not essentially affect theaffinity of the SDAB molecule (e.g. does not reduce said affinity bymore than 90%, preferably not by more than 50%, and by no more than 10%,as determined by a suitable assay, such as those described in theExamples below); and/or (4) does not affect any of the other desiredproperties of the SDAB molecule. Suitable PEG-groups and methods forattaching them, either specifically or non-specifically, will be clearto the skilled person.

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the SDAB molecule.

Formulations

A formulation of an SDAB molecule, e.g., nanobody molecule, includes anSDAB molecule, a compound that can serve as a cryoprotectant, and abuffer. The pH of the formulation is generally pH 5.5±7.0. In someembodiments, a formulation is stored as a liquid. In other embodiments,a formulation is prepared as a liquid and then is dried, e.g., bylyophilization or spray-drying, prior to storage. A dried formulationcan be used as a dry compound, e.g., as an aerosol or powder, orreconstituted to its original or another concentration, e.g., usingwater, a buffer, or other appropriate liquid.

The SDAB molecule purification process is designed to permit transfer ofthe an SDAB molecule into a formulation suitable for long-term storageas a frozen liquid and subsequently for freeze-drying (e.g., using ahistidine/sucrose formulation). The formulation is lyophilized with theprotein at a specific concentration. The lyophilized formulation canthen be reconstituted as needed with a suitable diluent (e.g., water) toresolubilize the original formulation components to a desiredconcentration, generally the same or higher concentration compared tothe concentration prior to lyophilization.

The lyophilized formulation may be reconstituted to produce aformulation that has a concentration that differs from the originalconcentration (i.e., before lyophilization), depending upon the amountof water or diluent added to the lyophilate relative to the volume ofliquid that was originally freeze-dried. Suitable formulations can beidentified by assaying one or more parameters of antibody integrity. Theassayed parameters are generally the percentage of HMW species or thepercentage of LMW species.

The percentage of HMW species or LMW species is determined either as apercentage of the total protein content in a formulation or as a changein the percentage increase over time (i.e., during storage). The totalpercentage of HMW species in an acceptable formulation is not greaterthan 10% HMW species after storage as a lyophilate or liquid at −20° C.to 40° C. (e.g., at −20° C. to 25° C., at −20° C. to 15° C., at 2° C. to8° C., at about 2° C., or at about 25° C.) for at least one year or notgreater than about 10% LMW species after storage as a lyophilate orliquid at −20° C. to 40° C. for at least one year. By “about” is meant±20% of a cited numerical value. Thus, “about 20° C.” means 16° C. to24° C.

Typically, the stability profile is less than 10% HMW/LMW at 2°-8° C.for a refrigerated product, and 25° C. for a room-temperature product.HMW species or LMW species are assayed in a formulation stored as alyophilate after the lyophilate is reconstituted. 40° C. is anaccelerated condition that is generally used for testing stability anddetermining stability for short-term exposures to non-storageconditions, e.g., as may occur during transfer of a product duringshipping.

When the assayed parameter is the percentage change in HMW species orLMW species, the percent of total protein in one or both species afterstorage is compared to the percent total protein in one or both speciesprior to storage (e.g., upon preparation of the formulation). Thedifference in the percentages is determined. In general, the change inthe percentage of protein in HMW species or LMW species in liquidformulations is not greater than 10%, e.g., not greater than about 8%,not greater than about 7%, not greater than about 6%, not greater thanabout 5%, not greater than about 4%, or not greater than about 3% afterstorage at 2° C.-8° C. or 25° C. for about eighteen to twenty-fourmonths. By “about” is meant ±20% of a cited numerical value, typically,within 10%, and more typically, within 5% of a given value or range ofvalues. Thus, about 10% means 8% to 12%. Formulations stored aslyophilized product generally have less than about 5%, less than about4%, less than about 3%, less than about 2%, or less than about 1% of HMWspecies or less than about 5%, less than about 4%, less than about 3%,or less than about 2%, or less than about 1% of LMW species afterreconstitution, or in liquid formulation, following storage at −30°C.-8° C. (e.g., 4° C., or −20° C.) for about six, nine, ten, twelve,fifteen, eighteen to twenty-four months.

Formulations of SDAB molecules (e.g., TNF-binding nanobody molecules)can be stored as a frozen liquid formulation or a lyophilate for, e.g.,at least six, nine, ten, twelve months, or at least two years, at leastthree years, at least four years, or at least five years. In oneexample, a TNF-binding nanobody molecule formulation contains 10 mMhistidine, 5% sucrose, 0.01% Polysorbate 80, 50 mg/mL TNF-bindingnanobody molecules, and has a pH of 6.0. In another example, theTNF-binding nanobody molecule formulation contains 20 mM histidine, 7.5%sucrose, 0.01% Polysorbate 80, 50 mg/mL TNF-binding nanobody molecules,and has a pH of 6.0. In another example, the formulation contains 20 mMhistidine, 10% sucrose, 0.02% Polysorbate 80, 100 mg/mL TNF-bindingnanobody molecule, and has a pH of 6.0. In another example, theformulation contains 10 mM histidine, 5% sucrose, 50 mg/mL TNF-bindingnanobody molecule, and has a pH of 6.0. In yet another example, theformulation contains 20 mM histidine, 10% sucrose, 100 mg/mL TNF-bindingnanobody, and has a pH of 6.0. In another example, the formulationcontains 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80,approximately 80 mg/mL TNF-binding nanobody molecule, and has a pH of6.0. In yet another example, the formulation contains 10 mM histidine,5% sucrose, 0.01% Polysorbate 80, 100 mM Arginine (base), 88 to 100mg/mL TNF-binding nanobody molecule, and has a pH of 5.8. In anotherexample, the formulation contains 10 mM histidine, 5% sucrose, 0.01%Polysorbate 80, 55 mM NaCl, 88 to 100 mg/mL TNF-binding nanobodymolecule, and has a pH of 6.1. In yet another example, the formulationcontains 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80, 55 mMArginine HCl, 88 to 100 mg/mL TNF-binding nanobody molecule, and has apH of 6.1. In another example, the formulation contains 10 mM histidine,5% sucrose, 0.01% Polysorbate 80, 100 mM Glycine, 88 to 100 mg/mLTNF-binding nanobody molecule, and has a pH of 6.0. In yet anotherexample, the formulation contains 10 mM histidine, 5% sucrose, 0.01%Polysorbate 80, 100 mM Methionine, 88 to 100 mg/mL TNF-binding nanobodymolecule, and has a pH of 6.0. In another example, the formulationcontains 10 mM histidine, 8% sucrose, 0.01% Polysorbate 80, 88 to 100mg/mL TNF-binding nanobody molecule, and has a pH of 6.0. In yet anotherexample, the formulation contains 10 mM histidine, 5% sucrose, 0.01%Polysorbate 80, 88 to 100 mg/mL TNF-binding nanobody molecule, and has apH of 6.0. In another example, the formulation contains 20 mM Histidine,5% Sucrose, 118 mg/mL TNF-binding nanobody molecule, and has a of pH6.0. In yet another example, the formulation contains 20 mM Tris, 5%Sucrose, 117 mg/mL TNF-binding nanobody molecule, has a pH of 7.2. Inanother example, the formulation contains 10 mM histidine, 5% sucrose,0.01% Polysorbate 80, approximately 80 mg/mL TNF-binding nanobodymolecule, and has a pH of 6.0. In yet another example, the formulationcontains 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80,approximately 50 mg/mL TNF-binding nanobody molecule, and has a pH of6.0. In one example, the formulation contains 10 mM histidine, 5%sucrose, 0.01% Tween-80, approximately 1 mg/mL TNF-binding nanobodymolecule, and has a pH of 5.5. In another example, the formulationcontains 10 mM histidine, 5% sucrose, 0.01% Tween-80, 150 mM arginineHCl, approximately 1 mg/mL TNF-binding nanobody molecule, and has a pHof 5.5. In yet another example, the formulation contains 10 mMhistidine, 5% sucrose, 0.01% Tween-80, 75 mM sodium chloride,approximately 1 mg/mL TNF-binding nanobody molecule, and has a pH of5.5. In one example, the formulation contains 10 mM histidine, 5%sucrose, 0.01% Tween-80, approximately 1 mg/mL TNF-binding nanobodymolecule, and has a pH of 6.0. In another example, the formulationcontains 10 mM histidine, 5% sucrose, 0.01% Tween-80, 150 mM arginineHCl, approximately 1 mg/mL TNF-binding nanobody, and has a pH of 6.0. Inyet another example, the formulation contains 10 mM histidine, 5%sucrose, 0.01% Tween-80, 75 mM sodium chloride, approximately 1 mg/mLTNF-binding nanobody molecule, and has a pH of 6.0. In one example, theformulation contains 10 mM histidine, 5% sucrose, 0.01% Tween-80,approximately 1 mg/mL TNF-binding nanobody molecule, and has a pH of6.5. In another example, the formulation contains 10 mM histidine, 5%sucrose, 0.01% Tween-80, 150 mM arginine HCl, approximately 1 mg/mLTNF-binding nanobody molecule, and has a pH of 6.5. In yet anotherexample, the formulation contains 10 mM histidine, 5% sucrose, 0.01%Tween-80, 75 mM sodium chloride, approximately 1 mg/mL TNF-bindingnanobody molecule, and has a pH of 6.5. In one example, the formulationcontains 10 mM histidine, 5% sucrose, 0.01% Tween-80, approximately 1mg/mL TNF-binding nanobody molecule, and has a pH of 7.0. In anotherexample, the formulation contains 10 mM histidine, 5% sucrose, 0.01%Tween-80, 150 mM arginine HCl, approximately 1 mg/mL TNF-bindingnanobody molecule, and has a pH of 7.0. In yet another example, theformulation contains 10 mM histidine, 5% sucrose, 0.01% Tween-80, 75 mMsodium chloride, approximately 1 mg/mL TNF-binding nanobody molecule,and has a pH of 7.0. In yet another example, the TNF-binding nanobodymolecule formulation contains 20 mM histidine, 7.5% sucrose, 0.01%Polysorbate 80, 250 mg/mL TNF-binding nanobody molecules, and has a pHof 6.0.

Additional details related to components of formulations and methods ofassaying the integrity of the SDAB molecule, e.g., the TNF-bindingnanobody molecule, in a formulation are provided infra.

SDAB molecule concentrations in formulations are generally between about0.1 mg/mL and about 350 mg/mL, e.g., 0.5 mg/mL to about 350 mg/mL, about0.5 mg/mL to about 300 mg/mL, about 0.5 mg/mL to about 250 mg/mL, about0.5 mg/mL to about 150 mg/mL, about 1 mg/ml to about 130 mg/mL, about 10mg/ml to about 130 mg/mL, about 50 mg/ml to about 120 mg/mL, about 80mg/ml to about 120 mg/mL, about 88 mg/ml to about 100 mg/mL or about 10mg/ml, about 25 mg/ml, about 50 mg/ml, about 80 mg/ml, about 100 mg/mL,about 130 mg/ml, about 150 mg/ml, about 200 mg/ml, about 250 mg/ml orabout 300 mg/ml. In the context of ranges, “about” means −20% of thelower-cited numerical value of the range and +20% of the upper-citednumerical value of the range. In the context of ranges, e.g., about 10mg/mL to about 100 mg/mL, this means, between 8 mg/mL to 120 mg/mL. Insome cases, SDAB molecule concentrations in formulations can be, forexample, between 0.1 mg/mL and 200 mg/mL, e.g., 0.5 mg/mL and 100 mg/mL,0.5 mg/mL and 1.0 mg/mL, 0.5 mg/mL and 45 mg/mL, 1 mg/mL and 10 mg/mL,10 mg/mL and 40 mg/mL, 10 mg/mL and 50 mg/mL, 50 mg/mL and 100 mg/mL,100 mg/mL and 200 mg/mL. Such SDAB molecule formulations can be used astherapeutic agents. Accordingly, the concentration of SDAB molecule in aformulation is sufficient to provide such dosages in a volume of theformulation that is tolerated by a subject being treated and isappropriate for the method of administration. In one non-limitingexample, to supply a high dosage subcutaneously, in which the volumelimitation is small (e.g., about 1 ml to 1.2 ml per injection), theconcentration of SDAB molecule is generally at least 100 mg/mL orgreater, e.g., 100 mg/mL to 500 mg/mL, 100 mg/mL to 250 mg/mL, or 100mg/mL to 150 mg/mL. Such high concentrations can be achieved, forexample, by reconstituting a lyophilized formulation in an appropriatevolume of diluent (e.g., sterile water for injection, buffered saline).In some cases, the reconstituted formulation has a concentration ofbetween about 100 mg/mL and 300 mg/mL (e.g., 100 mg/mL, 125 mg/mL, 150mg/mL, 175 mg/mL, 200 mg/mL, 250 mg/mL, 275 mg/mL, 300 mg/mL). Highconcentrations, for example up to 250 mg/mL, can be used for long termstorage, e.g., frozen storage of large preparations of the SDABmolecule.

For delivery via inhalation, the formulation is generally somewhatconcentrated (e.g., between about 100 mg/mL and 500 mg/mL) so as toprovide a sufficient dose in a limited volume of aerosol forinspiration. In some cases, low concentrations (e.g., between about 0.05mg/mL and 1 mg/mL) are used. Methods are known in the art to adapt thedosage delivered to the method of delivery, e.g., a jet nebulizer or ametered aerosol.

Buffers and Cryoprotectants

The pH of a formulation as described herein is generally between aboutpH 5.0 to about 7.0, for example, about pH 5.5 to about 6.5, about pH5.5 to about 6.0, about pH 6.0 to about 6.5, pH 5.5, pH 6.0, or pH 6.5.In general, a buffer that can maintain a solution at pH 5.5 to 6.5 isused to prepare a formulation, e.g., a buffer having a pKA of about 6.0.Suitable buffers include, without limitation, histidine buffer, TRIS,2-(N-morpholino)ethanesulfonic acid (MES), cacodylate, phosphate,acetate, succinate, and citrate. The concentration of the buffer isbetween about 4 mM and about 60 mM, e.g., about 5 mM to about 25 mM, forexample, histidine is generally used at a concentration of up to 60 mM.In some cases, histidine buffer is used at a concentration of about 5mM, about 10 mM or about 20 mM. In other cases, acetate or succinatebuffer is used at a concentration of about 5 mM or about 10 mM.

Cryoprotectants are known in the art and include, e.g., sucrose,trehalose, and glycerol. A cryoprotectant exhibiting low toxicity inbiological systems is generally used. The cryoprotectant is included inthe formulation at a concentration of about 0.5% to 15%, about 0.5% to2%, about 2% to 5%, about 5% to 10%, about 10% to 15%, and about 5%(weight/volume).

Histidine buffer, which can be used as a buffer in an TNF-bindingnanobody formulation, may have cryoprotectant properties. In someembodiments of the invention, a histidine buffer is used in conjunctionwith a cryoprotectant such as a sugar, e.g., sucrose. A formulation ofthe invention can specifically exclude the use of histidine in anysubstantial amount, e.g., neither the buffer nor the cryoprotectantcomponent of the formulation is a histidine.

The viscosity of a formulation is generally one that is compatible withthe route of administration of the formulation. In some embodiments, theviscosity of the formulation is between 1 cP and 4 cP, e.g., about 2 cPto 3.5 cP. In other embodiments, the viscosity of the formulation isbetween about 5 cP and about 40 cP. In specific embodiments, theviscosity of the formulation is about 1 cP, 2 cP, 2.4 cP to 2.8 cP, 3cP, 3.1 cP to 3.2 cP, 4 cP, 5 cP, 10 cP, 15 cP, 20 cP, 25 cP, 30 cP, 35cP, or 40 cP.

Surfactants

In certain embodiments, a surfactant is included in the formulation.Examples of surfactants include, without limitation, nonionicsurfactants such as polysorbates (e.g., polysorbate-20, polysorbate-40,polysorbate-60, polysorbate-65, polysorbate-80, or polysorbate-85);Triton™; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodiumoctyl glycoside; lauryl-sulfobetaine, myristyl-sulfobetaine,linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine,myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine,linoleyl-betaine, myristyl-betaine, cetyl-betaine,lauroamidopropyl-betaine, cocamidopropyl-betaine,linoleamidopropyl-betaine, myristamidopropyl-betaine,palmidopropyl-betaine, isostearamidopropyl-betaine (e.g.lauroamidopropyl), myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the Monaquat™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol e.g., poloxamers (e.g., poloxamer 188).

The amount of surfactant added is such that it reduces aggregation ofthe reconstituted protein to an acceptable level as assayed using, e.g.,SEC-HPLC of HMW species or LMW species, and minimizes the formation ofparticulates after reconstitution of a lyophilate of an TNF-bindingnanobody formulation. The addition of surfactant has also been shown toreduce the reconstitution time of a lyophilized formulation ofTNF-binding antibodies, and aid in de-gassing the solution. For example,the surfactant can be present in the formulation (liquid or prior tolyophilization) in an amount from about 0.001% to 0.6%, e.g., from about0.005% to 0.05%, about 0.005% to about 0.2%, and about 0.01% to 0.2%.

Additions to Formulations

Formulations are stored as sterile solutions or sterile lyophilates.Prevention of the action of microorganisms in formulations can also beachieved by including at least one antibacterial and/or antifungal agentin a formulation, for example, parabens, chlorobutanol, phenol, ascorbicacid, thimerosal, and the like. In some cases, a lyophilate isreconstituted with bacteriostatic water (e.g., water containing 0.9%benzyl alcohol). Considerations for the inclusion of a preservative in aformulation are known in the art as are methods of identifyingpreservatives that are compatible with a specific formulation and methodof delivery (e.g., see Gupta, et al. (2003), AAPS Pharm. Sci. 5:article8, p. 1-9).

In some cases, the formulation is isotonic. In general, any componentknown in the art that contributes to the solution osmolarity/tonicitycan be added to a formulation (e.g., salts, sugars, polyalcohols, or acombination thereof). Isotonicity is generally achieved using either acomponent of a basic formulation (such as sucrose) in an isotonicconcentration or by adding an additional component such as, a sugar, apolyalcohol such as manitol or sorbitol, or a salt such as sodiumchloride.

In some cases, a salt is used in an TNF-binding nanobody formulation,e.g., to achieve isotonicity or to increase the integrity of theTNF-binding nanobody of the formulation. Salts suitable for use arediscussed, supra. The salt concentration can be from 0 mM to about 300mM.

In certain cases, the formulation is prepared with Tween (e.g., Tween®20, Tween® 80) to decrease interfacial degradation. The Tweenconcentration can be from about 0.001% to about 0.05%. In one example,Tween 80 is used at a concentration of 0.01% in the formulation.

In certain other cases, the formulation is prepared with arginine. Thearginine concentration in the formulation can be from about 0.01% toabout 5%. In one example, arginine is used at a concentration of 2% inthe formulation. In some cases both Tween and arginine are added to theTNF-binding formulations described herein.

In yet other cases, the formulation may be prepared with at least oneof: sorbitol, glycine, methionine, or sodium chloride. If sorbitol isincluded in the formulation, it can be added to a concentration ofbetween about 1% and about 10%. In one example, sorbitol is found in theformulation at a concentration of 5%. If glycine is included in theformulation, it can be added to a concentration of between about 0.1% toabout 2%. In one example, glycine is found in the formulation at aconcentration of 1%. If methionine is included in the formulation, itcan be added to a concentration of between about 5 mM and about 150 mM.In one example, methionine is added to the formulation at aconcentration of 100 mM. In another example, methionine is added to theformulation at a concentration of about 10 mM, about 20 mM or about 70mM. If sodium chloride is included in the formulation, it can be addedto a concentration of between about 5 mM and about 100 mM. In oneexample, sodium chloride is added to the formulation at a concentrationof 55 mM.

Storage and Preparation Methods

Freezing

In some cases, formulations containing antibodies are frozen forstorage. Accordingly, it is desirable that the formulation be relativelystable under such conditions, including, under freeze-thaw cycles. Onemethod of determining the suitability of a formulation is to subject asample formulation to at least two, e.g., three, four, five, eight, ten,or more cycles of freezing (at, for example −20° C. or −80° C.) andthawing (for example by fast thaw in a 37° C. water bath or slow thaw at2°-8° C.), determining the amount of LMW species and/or HMW species thataccumulate after the freeze-thaw cycles and comparing it to the amountof LMW species or HMW species present in the sample prior to thefreeze-thaw procedure. An increase in the LMW or HMW species indicatesdecreased stability.

Lyophilization

Formulations can be stored after lyophilization. Therefore, testing aformulation for the stability of the protein component of theformulation after lyophilization is useful for determining thesuitability of a formulation. The method is similar to that described,supra, for freezing, except that the sample formulation is lyophilizedinstead of frozen, reconstituted to its original volume, and tested forthe presence of LMW species and/or HMW species. The lyophilized sampleformulation is compared to a corresponding sample formulation that wasnot lyophilized. An increase in LMW or HMW species in the lyophilizedsample compared to the corresponding sample indicates decreasedstability in the lyophilized sample.

In general, a lyophilization protocol includes loading a sample into alyophilizer, a pre-cooling period, freezing, vacuum initiation, rampingto the primary drying temperature, primary drying, ramping to thesecondary drying temperature, secondary drying, and stoppering thesample. Additional parameters that can be selected for a lyophilizationprotocol include vacuum (e.g., in microns) and condenser temperature.Suitable ramp rates for temperature are between about 0.1° C./min. to 2°C./min., for example 0.1° C./min. to 1.0° C./min., 0.1° C./min. to 0.5°C./min., 0.2° C./min. to 0.5° C./min., 0.1° C./min., 0.2° C./min., 0.3°C./min., 0.4° C./min., 0.5° C./min., 0.6° C./min., 0.7° C./min., 0.8°C./min., 0.9° C./min., and 1.0° C./min. Suitable shelf temperaturesduring freezing for a lyophilization cycle are generally from about −55°C. to −5° C., −25° C. to −5° C., −20° C. to −5° C., −15° C. to −5° C.,−10 C to −5° C., −10° C., −11° C., −12° C., −13° C., −14° C., −15° C.,−16° C., −17° C., −18° C., −19° C., −20° C., −21° C., −22° C., −23° C.,−24° C., or −25° C. Shelf temperatures can be different for primarydrying and secondary drying, for example, primary drying can beperformed at a lower temperature than secondary drying. In anon-limiting example, primary drying can be executed at 0° C. andsecondary drying at 25° C.

In some cases, an annealing protocol is used during freezing and priorto vacuum initiation. In such cases, the annealing time must be selectedand the temperature is generally above the glass transition temperatureof the composition. In general, the annealing time is about 2 to 15hours, about 3 to 12 hours, about 2 to 10 hours, about 3 to 5 hours,about 3 to 4 hours, about 2 hours, about 3 hours, about 5 hours, about 8hours, about 10 hours, about 12 hours, or about 15 hours. Thetemperature for annealing is generally from about −35° C. to about −5°C., for example from about −25° C. to about −8° C., about −20° C. toabout −10° C., about −25° C., about −20° C., about −15° C., about 0° C.,or about −5° C. In some cases, the annealing temperature is generallyfrom −35° C. to 0° C., for example from −25° C. to −8° C., −20° C. to−10° C., −25° C., −20° C., −15° C., 0° C.

The stability of the formulations described herein can be tested using avariety of lyophilization parameters including: the primary drying shelftemperatures from −25° C. to 30° C., and secondary drying durations of 2hours to 9 hours at 0° to 30° C.

In one non-limiting example, a formulation of 10 mM histidine, 5%sucrose, 0.01% Polysorbate 80, pH 6.0, at a protein concentration of 50mg/mL TNF-binding nanobody was formulated in bulk and lyophilized. Afterlyophilization, the product is reconstituted with approximately half thefill volume to deliver protein at 100 mg/mL. The TNF antibody wasdemonstrated to be robust after lyophilization to extremes in producttemperature. The stability profile upon storage at 50° C. for four weekswas identical for material that had been prepared using a variety offreeze-drying cycles (e.g., see FIGS. 16-20), some of which had nearly10° C. differences in product temperature during primary drying (e.g.,FIG. 13). In general, a lyophilization cycle can run from 10 hours to100 hours, e.g., 20 hours to 80 hours, 30 hours to 60 hours, 40 hours to60 hours, 45 hours to 50 hours, 50 hours to 65 hours.

Non-limiting examples of the temperature range for storage of anantibody formulation are about −20° C. to about 50° C., e.g., about −15°C. to about 30° C., about −15° C. to about 20° C., about 5° C. to about25° C., about 5° C. to about 20° C., about 5° C. to about 15° C., about2° C. to about 12° C., about 2° C. to about 10° C., about 2° C. to about8° C., about 2° C. to about 6° C., or about 2° C., 3° C., 4° C., 5° C.,6° C., 7° C., 8° C., 10° C., 15° C., 25° C., or 30° C. Notwithstandingthe storage temperatures, in certain cases, samples are stable undertemperature changes that may transiently occur during storage andtransportation conditions that can be anticipated for such compositions.

Spray-Drying

In some cases, a formulation is spray-dried and then stored.Spray-drying is conducted using methods known in the art, and can bemodified to use liquid or frozen spray-drying (e.g., using methods suchas those from Niro Inc. (Madison, Wis.), Upperton Particle Technologies(Nottingham, England), or Buchi (Brinkman Instruments Inc., Westbury,N.Y.), or U.S. Patent Publ. Nos. 20030072718 and 20030082276).

Determination of SDAB Molecule Integrity

The accumulation of LMW species and HMW species are useful measures ofantibody stability. Accumulation of either LMW or HMW in a formulationis indicative of instability of a protein stored as part of theformulation. Size exclusion chromatography with HPLC can be used todetermine the presence of LMW and HMW species. Suitable systems for suchmeasurements are known in the art, e.g., HPLC systems (Waters, Milford,Mass.). Other systems known in the art can be used to evaluate theintegrity of antibody in a formulation, for example, SDS-PAGE (tomonitor HMW and LMW species), bioassays of antibody activity,enzyme-linked immunosorbent assay, ability to bind purified targetprotein (e.g., TNFα), and cation exchange-HPLC (CEX-HPLC; to detectvariants and monitor surface charge). In one example, a bioassay is acell-based assay in which inhibition of TNFα-dependent activity isexamined in the presence of different concentrations of formulatednanobody molecule to demonstrate biological activity.

Articles of Manufacture

The present application also provides an article of manufacture thatincludes a formulation as described herein and provides instructions foruse of the formulation.

Formulations to be used for administration to a subject, e.g., as apharmaceutical, must be sterile. This is accomplished using methodsknown in the art, e.g., by filtration through sterile filtrationmembranes, prior to, or following, formulation of a liquid orlyophilization and reconstitution. Alternatively, when it will notdamage structure, components of the formulation can be sterilized byautoclaving and then combined with filter or radiation sterilizedcomponents to produce the formulation.

The pharmaceutical formulation can be administered with a transcutaneousdelivery device, such as a syringe, including a hypodermic ormultichamber syringe. In one embodiment, the device is a prefilledsyringe with attached or integral needle. In other embodiments, thedevice is a prefilled syringe not having a needle attached. The needlecan be packaged with the prefilled syringe. In one embodiment, thedevice is an auto-injection device, e.g., an auto-injector syringe. Inanother embodiment the injection device is a pen-injector. In yetanother embodiment, the syringe is a staked needle syringe, luer locksyringe, or luer slip syringe. Other suitable delivery devices includestents, catheters, microneedles, and implantable controlled releasedevices. The composition can be administered intravenously with standardIV equipment, including, e.g., IV tubings, with or without in-linefilters.

In certain embodiments, a syringe is suitable for use with anautoinjector device. For example, the autoinjector device can include asingle vial system, such as a pen-injector device for delivery of asolution. Such devices are commercially available from manufacturerssuch as BD Pens, BD Autojector®, Humaject®, NovoPen®, B-D®Pen, AutoPen®,and OptiPen®, GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen®,Roferon Pen®, Biojector®, Iject®, J-tip Needle-Free Injector®, DosePro®,Medi-Ject®, e.g., as made or developed by Becton Dickensen (FranklinLakes, N.J.), Ypsomed (Burgdorf, Switzerland, www.ypsomed.com; Bioject,Portland, Oreg.; National Medical Products, Weston Medical(Peterborough, UK), Medi-Ject Corp (Minneapolis, Minn.), and Zogenix,Inc, Emeryville, Calif. Recognized devices comprising a dual vial systeminclude those pen-injector systems for reconstituting a lyophilized drugin a cartridge for delivery of the reconstituted solution such as theHumatroPen®.

The article of manufacture can include a container suitable forcontaining the formulation. A suitable container can be, withoutlimitation, a device, bottle, vial, syringe, test tube, nebulizer (e.g.,ultrasonic or vibrating mesh nebulizers), i.v. solution bag, or inhaler(e.g., a metered dose inhaler (MDI) or dry powder inhaler (DPI)). Thecontainer can be formed of any suitable material such as glass, metal,or a plastic such as polycarbonate, polystyrene, or polypropylene. Forexample, the container (e.g., syringe or vial) can be formed out ofglass, plastic, a cyclic olefin copolymer, or a cyclic olefin polymer.Optionally, the container (e.g., syringe or vial) has a stopper, e.g., arubber stopper. Specific embodiments of containers for storing thepresent formulations include: (i) liquid in a glass vial with rubberstopper; (ii) liquid in a glass prefillable syringe with rubber plunger;and (iii) liquid in a prefillable polymeric syringe, for example cyclicolefin copolymer (COC), or cyclic olefin polymer (COP), with rubberplunger.

In general, the container is of a material that does not adsorbsignificant amounts of protein from the formulation and is not reactivewith components of the formulation.

In some embodiments, the container is a clear glass vial with a stopper,e.g., a West 4432/50 1319 siliconized gray stopper or a West 4023Durafluor stopper. In some embodiments, the container is a syringe. Inspecific embodiments, the formulation comprises 100 mg/mL of theTNF-binding nanobody, 20 mM histidine, 7.5% sucrose, 0.01%polysorbate-80, pH 6.0 in a pre-filled syringe. In another embodiment,the formulation comprises about 10 mg/mL, about 100 mg/mL of theTNF-binding nanobody, 20 mM histidine, 7.5% sucrose, 0.01%polysorbate-80, pH 6 in a prefillable cyclic olefin syringe and a West4432/50 siliconized gray rubber plunger. In other embodiments, theformulations include about 10 mg/mL, about 50 mg/mL, about 100 mg/mL ofthe TNF-binding nanobody, 20 mM histidine, 7.5% sucrose, 0.01%polysorbate-80, pH 6 in a prefillable glass syringe and a West 4432/50siliconized gray rubber plunger or West 4023/50 Dalkyo Fluorotec/B2coated rubber plunger.

The articles of manufacture described herein can further include apackaging material. The packaging material provides, in addition to theinformation for use or administration, e.g., information required by aregulatory agency regarding conditions under which the product can beused. For example, the packaging material can provide instructions tothe patient on how to inject a pre-filled syringe containing theformulations described herein, or how to reconstitute the lyophilizedformulation in an aqueous diluent to form a solution within a specifiedperiod, e.g., over a period of 2-24 hours or greater. The presentlyclaimed formulations are useful for human pharmaceutical product use.

In certain embodiments, the formulations can be administered asnebulizers. Examples of nebulizers include, in non-limiting examples,jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers.These classes use different methods to create an aerosol from a liquid.In general, any aerosol-generating device that can maintain theintegrity of the protein in these formulations is suitable for deliveryof formulations as described herein.

In other embodiments, the pharmaceutical compositions can beadministered with medical devices. For example, pharmaceuticalcompositions can be administered with a needleless hypodermic injectiondevice, such as the devices disclosed in U.S. Pat. Nos. 5,399,163,5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.Examples of well-known implants and modules include: U.S. Pat. No.4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicants throughthe skin; U.S. Pat. No. 4,447,233, which discloses a medication infusionpump for delivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. The therapeutic composition can also be in theform of a biodegradable or nonbiodegradable sustained releaseformulation for subcutaneous or intramuscular administration. See, e.g.,U.S. Pat. Nos. 3,773,919 and 4,767,628 and PCT Application No. WO94/15587. Continuous administration can also be achieved using animplantable or external pump. The administration can also be conductedintermittently, e.g., single daily injection, or continuously at a lowdose, e.g., sustained release formulation. The delivery device can bemodified to be optimally suited for administration of the SDAB molecule.For example, a syringe can be siliconized to an extent that is optimalfor storage and delivery of the SDAB molecule. Of course, many othersuch implants, delivery systems, and modules are also known. Theinvention also features a device for administering a first and secondagent. The device can include, e.g., one or more housings for storingpharmaceutical preparations, and can be configured to deliver unit dosesof the first and second agent. The first and second agents can be storedin the same or separate compartments. For example, the device cancombine the agents prior to administration. It is also possible to usedifferent devices to administer the first and second agent.

Administration and Method of Treatment

The formulations of the invention be administered to a subject (e.g., ahuman subject) alone or combination with a second agent, e.g., a secondtherapeutically or pharmacologically active agent, to treat or prevent(e.g., reduce or ameliorate one or more symptoms associated with) a TNFαassociated disorder, e.g., inflammatory or autoimmune disorders. Theterm “treating” refers to administering a therapy in an amount, manner,and/or mode effective to improve a condition, symptom, or parameterassociated with a disorder or to prevent progression of a disorder, toeither a statistically significant degree or to a degree detectable toone skilled in the art. An effective amount, manner, or mode can varydepending on the subject and may be tailored to the subject.

Non-limiting examples of immune disorders that can be treated include,but are not limited to, autoimmune disorders, e.g., arthritis (includingrheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,psoriatic arthritis, lupus-associated arthritis or ankylosingspondylitis), scleroderma, systemic lupus erythematosis, Sjogren'ssyndrome, vasculitis, multiple sclerosis, autoimmune thyroiditis,dermatitis (including atopic dermatitis and eczematous dermatitis),myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease,colitis, diabetes mellitus (type I); inflammatory conditions of, e.g.,the skin (e.g., psoriasis); acute inflammatory conditions (e.g.,endotoxemia, sepsis and septicaemia, toxic shock syndrome and infectiousdisease); transplant rejection and allergy. In one embodiment, the TNFαassociated disorder is, an arthritic disorder, e.g., a disorder chosenfrom one or more of rheumatoid arthritis, juvenile rheumatoid arthritis(RA) (e.g., moderate to severe rheumatoid arthritis), osteoarthritis,psoriatic arthritis, or ankylosing spondylitis, polyarticular juvenileidiopathic arthritis (JIA); or psoriasis, ulcerative colitis, Crohn'sdisease, inflammatory bowel disease, and/or multiple sclerosis.

In certain embodiments, the formulations include a second therapeuticagent. For example, for TNF-nanobodies, the second agent may be ananti-TNF antibody or TNF binding fragment thereof, wherein the secondTNF antibody has a different epitope specificity than the TNF-bindingSDAB molecule of the formulation. Other non-limiting examples of agentsthat can be co-formulated with TNF-binding SDAB include, for example, acytokine inhibitor, a growth factor inhibitor, an immunosuppressant, ananti-inflammatory agent, a metabolic inhibitor, an enzyme inhibitor, acytotoxic agent, and a cytostatic agent. In one embodiment, theadditional agent is a standard treatment for arthritis, including, butnot limited to, non-steroidal anti-inflammatory agents (NSAIDs);corticosteroids, including prednisolone, prednisone, cortisone, andtriamcinolone; and disease modifying anti-rheumatic drugs (DMARDs), suchas methotrexate, hydroxychloroquine (Plaquenil) and sulfasalazine,leflunomide (Arava®), tumor necrosis factor inhibitors, includingetanercept (Enbrel®), infliximab (Remicade®) (with or withoutmethotrexate), and adalimumab (Humira®), anti-CD20 antibody (e.g.,Rituxan®), soluble interleukin-1 receptor, such as anakinra (Kineret),gold, minocycline (Minocin®), penicillamine, and cytotoxic agents,including azathioprine, cyclophosphamide, and cyclosporine. Suchcombination therapies may advantageously utilize lower dosages of theadministered therapeutic agents, thus avoiding possible toxicities orcomplications associated with the various monotherapies.

The formulations of the invention can be in the form of a liquidsolution (e.g., injectable and infusible solutions). Such compositionscan be administered by a parenteral mode (e.g., subcutaneous,intraperitoneal, or intramuscular injection), or by inhalation. Thephrases “parenteral administration” and “administered parenterally” asused herein mean modes of administration other than enteral and topicaladministration, usually by injection, and include, subcutaneous orintramuscular administration, as well as intravenous, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcuticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion. In one embodiment, the formulationsdescribed herein are administered subcutaneously.

Pharmaceutical formulations are sterile and stable under the conditionsof manufacture and storage. A pharmaceutical composition can also betested to insure it meets regulatory and industry standards foradministration.

A pharmaceutical formulation can be formulated as a solution,microemulsion, dispersion, liposome, or other ordered structure suitableto high protein concentration. Sterile injectable solutions can beprepared by incorporating an agent described herein in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating anagent described herein into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

In some embodiments, parameters that describe the formulations, e.g.,parameters that may appear on the product label, are characterized. Suchparameters include, e.g., color (typically colorless to slightly yellow,or colorless to yellow), clarity (typically clear to slightlyopalescent, or clear to opalescent), and viscosity (typically betweenabout 1 to 5 cP when measured at ambient temperature, such as at 20° C.to 30° C.). Such parameters can be measured by methods known in the art.For example, clarity can be measured using commercially availableopalescence standards (available from, e.g., Hach Company, Loveland,Colo. 80539).

EXAMPLES

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstructed as limiting the scope or content of the invention in anyway.

Example 1 Stability of High Concentration Lyophilized Formulation ofATN-103 (6 Months Duration)

One method of storing an antibody to be used for, e.g., therapeuticapplications, is as a dried powder prepared by lyophilization.Accordingly, the long-term stability of a lyophilized TNF-bindingformulation was studied.

Briefly, a formulation containing a humanized TNF-binding nanobody (50mg/ml), 10 mM histidine, 5% sucrose (weight/volume), 0.01% Polysorbate80, pH 6.0, was prepared by sterile filtration and was dispensed into a5 ml depyrogenated glass tubing vial, and then lyophilized. Theformulation was stored at 4° C., 25° C., or 40° C. for one month, threemonths, and six months, then reconstituted in sterile water (USP) tobring the reconstituted formulation such that the formulation was 100mg/ml TNF-binding nanobody, 20 mM histidine, 10% sucrose, 0.02%Polysorbate 80, pH 6.0.

The stability of the high concentration liquid was assessed bybiological activity, Human Serum Albumin (HSA) binding, percentage ofHMW and percentage of LMW by SE-HPLC, percentage of TNF-binding nanobodyand percentage of non-product impurity by SDS-CE, and CEX-HPLCassessment of relative retention time and comparability of elutionprofile to TNF-binding nanobody reference standard.

The lyophilized TNF-binding nanobody formulations were assayed forbiological activity using an assay disclosed in WO 2006/122786. FIG. 1illustrates the data from such a set of bioassays. The data wereexpressed as units per milligram. Samples were about 5±5.5×10⁶ U/mgprior to storage and were about 4.5±5.5×10⁶ U/mg after incubation.Overall, there was no substantial change in the amount of bioactivityafter six months of storage in any of the samples. Thus, the formulationis, as determined by biological activity, suitable for storage of thelyophilized formulation for at least six months.

The lyophilized TNF-binding nanobody formulations were also assayed forHuman Serum Albumin (HSA) binding activity. FIG. 2 illustrates the datafrom such a set of binding assays. The initial binding activity of theformulation was about 100% of the reference sample and did not changesubstantially for any of the samples over the six-month period oftesting. Thus, the formulation is, as determined by HSA bindingactivity, suitable for storage of the lyophilized formulation for atleast six months.

The percentage of HMW species was assayed using SE-HPLC. The percentageof HMW species in the formulation before lyophilization andreconstitution was about 0.1% of the total protein in the formulationand was also between about 0.1%-0.2% in all samples stored at 4° C. and25° C. (FIG. 3). After six months of storage at 40° C., the formulationswere about 0.35% HMW species (FIG. 3). Thus, there was no substantialincrease in the level of HMW species in samples stored at 4° C. and 25°C. for six months.

The percentage of LMW species was assayed using SE-HPLC. The percentageof LMW species in the formulation was below limit of detection (i.e.0.0%) at temperatures of 4° C., 25° C. and 40° C. for up to six months.

The percentage of TNF-binding nanobody was assayed using SDS-CE. Theinitial percentage of TNF-binding nanobody in the formulation was about100% and did not change substantially for any of the samples over thesix-month period of testing (FIG. 4).

The percentage of non-product impurity was assayed using SDS-CE.Negligible non-product impurity was observed by SDS-CE for formulationat temperatures of 4° C., 25° C. and 40° C. for up to six months.

The lyophilized TNF-binding nanobody formulations were also tested foridentity using CEX-HPLC. The elution profile for the formulation wascomparable to reference standard at temperatures of 4° C., 25° C. and40° C. for up to six months. The relative retention time of designatedpeak was unchanged at 1.00 standard at temperatures of 4° C., 25° C. and40° C. for up to six months.

The effect of addition of Polysorbate-80 on reconstitution propertiesfor lyophilized TNF-binding nanobody formulation was tested as well. Theaddition of polysorbate 80 to the lyophilized product improves thequality of the product by improving the appearance and dissolution ofthe lyophilized powder as can be seen in the table below.

TABLE 1 With Polysorbate-80 Without Polysorbate-80 Recon Time 2 min, 39sec 3 min, 16 sec Clear Time Immediate <5 min Foaming Little foamSlightly more foam Bubble dissapation Immediate <3 min

The data described herein show limited changes in degradation productsas a function of storage time at various temperatures.

Example 2 Robustness of the TNF-Binding Nanobody Formulation toLyophilization

In addition to formulation lyophilized by applying the targetlyophilization cycle (Example 1), two additional lots of drug productwere prepared by applying two additional “robustness” lyophilizationcycles, to the same formulation. The two “robustness” lyophilizationcycles mimic significant process deviations that could occur in amanufacturing setting. The same drug product formulation was used in therobustness study as in the target (control) lyophilization cycle study:10 mM Histidine, 5% Sucrose, 0.01% Polysorbate 80, 50 mg/mL TNF-bindingnanobody, at pH 6.0. Upon reconstitution (using reconstitution diluentvolume approximately half that of the filled product prior tolyophilization) the ATN-103 formulation is as follows: 20 mM Histidine,10% Sucrose, 0.02% Polysorbate 80, 100 mg/mL TNF-binding nanobody, at pH6.0.

The two robustness lyophilization cycles that mimic significant processdeviations are termed “high moisture” and “aggressive”. FIG. 5demonstrates the formulation subjected to the robustness lyophilizationcycles shows comparable stability to that of the target (control) cycle.The lyophilization robustness formulation vials were placed onaccelerated stability side by side with the control lyophilizationcycle, and analyzed by SE-HPLC.

These data demonstrate that the ATN-103 lyophilized formulation isrobust to significant process deviations without product impact.

The percentage of LMW species for formulation subject to control androbustness lyophilization cycles was assayed using SE-HPLC. Thepercentage of LMW species by SE-HPLC for lyophilized TNF-bindingnanobody was below limit of detection (i.e. 0.0%) at t₀ and 50° C. forup to one month for all three cycles.

Lyophilization Practices

In all runs, an aluminum foil shield in front of the door and a shelfheight of 63 mm was used to minimize radiation within the lyophilizer.In all runs, one tray was entirely filled to maintain a consistent loadon the lyophilizer. Stoppers were autoclaved and dried for all proteinvials. All vials for protein samples were rinsed with de-ionized waterand depyrogenated. Vials and stoppers that were used to fill theremainder of the tray were untreated.

Vials seeded with the TNF-binding nanobody formulation were preparedaseptically in a biosafety cabinet at a target of 160 mg/vial. Vials forstability studies were filled with 3.2 ml of fresh formulation prior toeach run (material that had not been previously lyophilized). Duringlyophilization, additional vials were filled with suitable buffers thatwere compatible with the target lyophilization cycle to maintain aconsistent load on the lyophilizer. Lyophilization was monitored throughthe use of thermocouples within the protein array.

Modulated Differential Scanning Calorimetry (mDSC)

All samples for mDSC were run in modulated mode with an amplitude of0.5° C. and a period of 100 seconds. For post-lyophilization powders,samples were heated at 2° C./min. to 150° C. All powder samples wereprepared using a nitrogen-purged glove box. For liquid samples, alltemperature ramps were performed at 0.5° C./min. and temperatures werematched to those utilized in the lyophilization cycles. The finalheating ramp was performed at 2° C./min. to magnify the glasstransition. Liquid samples were prepared on the laboratory bench.

Moisture Analysis

Karl Fischer titration was used to assay moisture in lyophilizedsamples. Lyophilized samples were reconstituted with 3 ml methanol.

Duplicate or triplicate injections of 500 μL were performed. A 1% waterstandard was injected post use as a suitability check.

Fourier Transform Infrared Spectroscopy (FTIR)

FTIR measured secondary structure of the antibody in the dry powderstate. A pellet containing approximately 1 mg of formulated, driedprotein dispersed within 300 mg KBr was pressed and scanned 200 times.After data collection, analysis involved spectral subtraction of sucroseplacebo, baseline correction, smoothing, second derivative, and areanormalization.

Stability

The stability of lyophilized antibody in formulations was assessed as afunction of storage time and temperature. Samples of lyophilizedTNF-binding nanobody were assayed post-lyophilization, after four weeksof storage at 2° C.-8° C. and after two weeks and four weeks of storageat 50° C. Refrigerated samples were stored in a walk-in refrigeratedcold room. High temperature samples were stored in a Lab Line ImperialIncubator set at 50° C. At the appropriate time points samples wereremoved from storage and allowed to warm up or cool down to roomtemperature before assaying.

Reconstitution and Visual Appearance

Vials of lyophilized formulations from both post-lyophilization analysisand storage stability analysis were visually inspected before, during,and after being reconstituted with 1.3 ml of sterile water forinjection. Vials were inspected in a light box against both a black anda white background for cake color, integrity, moisture, particulates,and defects before reconstituting. After visually inspecting thelyophilized cake, the cap and crimp seal were removed from the vialusing a de-crimper. The stopper was removed and the sterile water forinjection was slowly dispensed into the vial using an appropriatepipette. The diluent was dispensed using a swirling motion to ensurefull wetting of the cake. Once the diluent was completely dispensed,timing of reconstitution was initiated with a standard laboratory timerand the vial was restoppered. Reconstitution was complete when the finalpiece of solid dissolved. Rolling the vial between one's handsfacilitated reconstitution. As the lyophilized cake was in the processof reconstituting, observations about the state of the dissolvingsolution such as clarity, bubbling, and foaming were recorded. Oncereconstitution was complete, the reconstitution time was recorded andthe vials were left on the bench for several minutes so that theresulting solution could settle and the majority of bubbles formedduring reconstitution could dissipate. The reconstituted solution wasthen inspected in a light box against both a black and a whitebackground for color, clarity, and particulates.

High Performance Size Exclusion Chromatography (SEC-HPLC)

Two microliters of neat samples of TNF-binding nanobody formulation wereinjected onto a G3000swxl column with a guard column (TosoHaas Part Nos.08541 and 08543). The mobile phase was phosphate buffered saline (PBS)with 250 mM sodium chloride added. The flow rate was 0.75 ml/min. andthe run time was 30 minutes. The ultraviolet absorbance was monitored ata wavelength of 280 nm. The chromatogram was integrated to separate themain TNF-binding nanobody peak from high and low molecular weightspecies using Waters Empower™ software.

Ultraviolet-Visible Absorbance Spectroscopy for ConcentrationDetermination (A₂₈₀)

Samples of the Formulation Having Antibody at a Concentration of 100mg/ml were diluted to approximately 0.5 mg/mL and 0.25 mg/mL by adding10 μl of sample to 1990 μl and 3990 μl of 10 mM histidine, 5% sucrose,pH 6.0, respectively. Two hundred microliters of the resulting solutionswere placed in individual wells in a 96-well microplate along with abuffer blank. The plate was read in a Spectramax® Plus plate reader forultraviolet absorbance at wavelengths of 280 nm and 320 nm. Subtractingthe 320 nm absorbance from the 280 nm absorbance and dividing by theextinction coefficient (1.405 mL/mg-cm) multiplied by the path length (1cm) determined protein concentrations of the solution in each well. Theappropriate dilution factor was applied, and an average proteinconcentration was determined.

Ultraviolet-Visible Absorbance Spectroscopy for Light Scatter (A₄₂₀)

Two hundred microliters of each TNF-binding nanobody sample to beanalyzed was aliquoted into individual wells on a 96-well microplate. Abuffer blank served as a control. The plate was read in a SpectramaxPlus plate reader for visible absorbance at a wavelength of 420 nm.

Cycle Development Strategy

A series of sequential steps (described below) were used to develop alyophilization cycle.

Critical Product Temperature Identification

The critical product temperature for an TNF-binding nanobody wasidentified by modulated Differential Scanning Calorimetry (mDSC). Thismethod is used to identify the glass transition temperature of thefrozen product (mDSC). A lyophilization cycle that maintains the productbelow this temperature during primary drying should yield an intact cakestructure. The lowest temperature suitable temperature was assumed to be−25° C., and so this temperature is generally included in proceduresdesigned to test conditions and formulations when developing aformulation and methods for lyophilization of an antibody as describedherein.

Lyophilization Cycle Execution

Based on the results from the studies described, supra, three differentlyophilization cycles were performed to examine three parameters ofinterest in developing a suitable lyophilization procedure for preparinga lyophilized formulation suitable for storage or other procedures. Thefirst parameter examined was control cycle, which repeats cycles fromprevious stability studies. All prior developmental stability cyclesutilized this cycle, so it served as a starting point for this analysis.

The second parameter tested was the impact of not performing thesecondary drying step, in order to generate lyophilized cakes with highresidual moisture content. This lyophilization cycle serves as anevaluation of the sensitivity of an TNF-binding nanobody formulation tohigh residual moisture content, and can be used in evaluation ofmanufacturing deviations during early clinical lots prior to theexecution of formal lyophilization robustness studies.

The third parameter tested was an aggressive cycle. Increasing theprimary drying temperature significantly above the control cycle setpoint can significantly increase the TNF-binding nanobody formulationproduct temperature during primary drying. This lyophilization cycleserves as an evaluation of the sensitivity of an TNF-binding nanobodyformulation to product temperature during lyophilization, and can beused in evaluation of manufacturing deviations during early clinicallots prior to the execution of formal lyophilization robustness studies.

Assessment of Lyophilization Cycles

The assessment of the selected lyophilization cycles on TNF-bindingnanobody formulations was split into two aspects: immediate comparisonbased on tests performed post-lyophilization, and potential longer-termimpact caused after incubation under accelerated conditions.

Critical Product Temperature Identification

The TNF-binding nanobody formulation product contained nearly 50%protein. As such, the protein was anticipated to dominate the physicalproperties of the frozen and lyophilized states. Prior tolyophilization, sub-ambient modulated Differential Scanning Calorimetry(mDSC) searched for the glass transition temperature of thefreeze-concentrated amorphous phase of the formulation. Based on datafrom the aggressive lyophilization development cycle, a producttemperature of −12° C. was selected as the critical temperature toremain below during lyophilization.

Example 3 Stability of High Concentration Liquid Formulation ofTNF-Binding Nanobody (6 Months Duration)

In some cases, it is desirable to store an TNF-binding nanobodyformulation in a liquid format. Accordingly, the long-term stability ofa liquid TNF-binding formulation containing a relatively highconcentration of TNF-binding nanobody was studied. Briefly, aformulation containing a humanized TNF-binding nanobody (approximately80 mg/mL), 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80, pH 6.0 wasprepared for storage by sterile filtering the formulation indepyrogenated stainless steel vessels. The formulation was stored at−20° C. or 4° C., for about three months and six months. The stabilityof the high concentration liquid was assessed by biological activity,Human Serum Albumin (HSA) binding, percentage of HMW and percentage ofLMW by SE-HPLC, percentage of ATN-103 and percentage of non-productimpurity by SDS-CE, and CEX-HPLC assessment of relative retention timeand comparability of elution profile to TNF-binding nanobody referencestandard.

A biological activity assay was used as stability parameter for the highconcentration liquid TNF-binding nanobody formulation. The assay wasconducted as described, supra, in Example 1. Samples were stored at −20°C. and 4° C. for about three months and six months. The data wereexpressed as units per milligram (FIG. 6). Samples were about 6×10⁶ U/mgprior to storage and were about 4.5−5×10⁶ U/mg after incubation. Thisreflects essentially no change in the bioactivity of the samples duringstorage. The variability in the values reflects the variability inherentin the assay. Because there is no decrease in the amount of biologicalactivity in the samples, these data provide further support for thesuitability of the formulation for storage of TNF-binding.

Yet another stability parameter was examined using the highconcentration liquid TNF-binding nanobody formulation: that of bindingactivity. In these experiments, the percentage of binding activity ofthe formulation was determined compared to a control after storage at−20° and 4° C. for six months. The assay specifically monitors thebinding affinity of the TNF-binding to Human Serum Albumin (HSA). Theinitial binding activity of the formulation was about 100% of thereference sample and did not change substantially for any of the samplesover the six-month period of testing (FIG. 7). Measured binding activitywas up to about 110% of the reference, which, given the error generallyobserved in this assay, reflects essentially no change in the bindingactivity of the samples over time, and there were no temperature-relatedtrends in binding results.

The percentage of HMW species was assayed using SEC-HPLC. The percentageof high molecular weight species in the high concentration liquidformulation before storage was between 0.1%-0.15% of the total proteinin the formulation and was about 0.1% in samples stored at −20° C., andabout 0.2% in samples stored at 4° C. up to six months storage (FIG. 8).Thus, there was no substantial increase in the level of HMW species insamples stored at −20° C. and 4° C. for at least six months.

The percentage of LMW species in the high concentration liquidTNF-binding nanobody formulation was also assayed in the TNF-bindingnanobody liquid formulation. The percentage of LMW species in theformulation was below limit of detection (i.e. 0.0%) at temperature of−20° C., and was about 0.1% in samples stored at 4° C. for up to sixmonths (FIG. 9). Thus, there was no substantial increase in the level ofLMW species in samples stored at −20° C. and 4° C. for at least sixmonths.

The percentage of LMW species was assayed using SE-HPLC. The percentageof LMW species in the high concentration liquid formulation was belowlimit of detection (i.e. 0.0%) at temperatures of 4° C., 25° C. and 40°C. for up to six months.

The percentage of TNF-binding nanobody was assayed using SDS-CE. Theinitial percentage of TNF-binding nanobody in the high concentrationliquid formulation was about 100% and did not change substantially forany of the samples over the six-month period of testing (FIG. 10).

The percentage of non-product impurity was assayed using SDS-CE.Negligible non-product impurity was observed by SDS-CE for liquid highconcentration TNF-binding nanobody formulation at temperatures of −20°C. and 4° C. for up to six months.

The high concentration liquid formulations were also tested for identityusing CEX-HPLC. CEX-HPLC is employed as a test of identity. The elutionprofile for TNF-binding of high concentration liquid formulation wascomparable to reference standard at temperatures of −20° C. and 4° C.for up to six months. The relative retention time of designated peak wasunchanged at 1.00 standard at temperatures of −20° C. and 4° C. for upto six months.

The data described herein show limited changes in degradation productsas a function of storage time at various temperatures.

Example 4 Stability of High Concentration Liquid Formulation ofTNF-Binding Nanobody in a Liquid Prefilled Syringe (12 Months Duration)

The stability of an TNF-binding nanobody high concentration liquidfilled into a prefilled syringe in the following formulation: 10 mMHistidine, 5% Sucrose, 0.01% Polysorbate 80, approximately 80 mg/mLTNF-binding nanobody, at pH 6.0 was assessed by percentage of HMW andpercentage of LMW by SE-HPLC and percentage of acidic and basic speciesby CEX-HPLC, and assessment of relative retention time and comparabilityof elution profile to TNF-binding nanobody reference standard. Theformulation was stored at 4° C. for twelve months, at 25° C. for threemonths, and at 40° C. for two months.

At the initial time point, there were about 0.7% HMW species. Aftertwelve months at, 4° C. there was a minimal increase to about 0.8% HMWspecies. After three months at 25° C., the HMW species increased toabout 1.8%. After two months at 40° C., the HMW species increased overtime to about 27% (FIG. 11).

At the initial time point, there were about 0.1% LMW species. Aftertwelve months at 4° C. there was a minimal increase to 0.25% LMWspecies. After three months at 25° C., there was a small increase toabout 0.5% LMW. After two months at 40° C., the degradation increasedover time to about 1.4% LMW species (FIG. 12).

At the initial time point, there were about 6% acidic species. Aftertwelve months at 4° C., there were about 7.5% acidic species. Afterthree months at 25° C., there were about 7.3% acidic species, with theacidic species increasing over time. After two months at 40° C., theacidic species increased over time to about 8.3% (FIG. 13).

At the initial time point, there were about 1.7% basic species. Aftertwelve months at 4° C., there were about 2.9% basic species. After threemonths at 25° C., there were about 2.9% basic species, with the basicspecies increasing over time. After two months at 40° C., the basicspecies increased over time to about 27% (FIG. 14).

The relative retention times and elution profiles of all samples werecomparable to TNF-binding nanobody reference standard.

The data show limited changes in degradation products as a function ofstorage time at 4° C. and 25° C., indicating the formulation is suitableas a liquid in a prefilled syringe. Some noticeable changes indegradation products were observed at 40° C., which is a stresscondition for a liquid.

Example 5 Stability of ATN-103 High Concentration Liquids—OtherFormulations (Identification of Other Stabilizing and DestabilizingExcipients)

In order to screen for possible excipients for an TNF-binding nanobodyliquid formulation, the stability of other high concentrationTNF-binding nanobody liquid formulations were examined. Supplementalwork was performed using various excipients to provide further stabilityand to make the formulation isotonic (suitable for injection in humansubjects). TNF-binding nanobody concentration is ranged from 88 mg/mL to100 mg/mL.

The formulations examined were:

1. 10 mM histidine, 5% sucrose, 0.01% polysorbate-80, 100 mM Arginine(base), pH 5.82. 10 mM histidine, 5% sucrose, 0.01% polysorbate-80, 55 mM NaCl, pH 6.13. 10 mM histidine, 5% sucrose, 0.01% polysorbate-80, 55 mM ArginineHCl, pH 6.14. 10 mM histidine, 5% sucrose, 0.01% polysorbate-80, 100 mM Glycine, pH6.05. 10 mM histidine, 5% sucrose, 0.01% polysorbate-80, 100 mM Methionine,pH 6.06. 10 mM histidine, 8% sucrose, 0.01% polysorbate-80, pH 6.0CTL: 10 mM histidine, 5% sucrose, 0.01% polysorbate-80, pH 6.0

The initial solution properties were analyzed for pH, osmolality,concentration, turbidity, and viscosity. All formulations resulted inisotonic solutions and showed acceptable clarity via A455 measurementand low viscosity (2.4 cP to 3.1 cP), showing prefilled syringe andauto-injector feasibility.

TABLE 2 Initial Solution Properties Formulation mOsm pH mg/mL TurbidityViscosity (cP) 1 356 5.82 98 <III 3.1 2 312 6.13 88 <III 2.6 3 303 6.1289 <III 2.6 4 305 6.05 88 <III 2.4 5 309 5.97 99 <III 2.8 6 306 6.03 88<III 2.6 CTL/0 197 6.01 100 <III 2.8

The stability of the high concentration liquid was assessed bypercentage of HMW and percentage of LMW by SE-HPLC. These materials wereplaced on stability at 5° C., 25° C. and 40° C. for 3 months. Data from40° C. 2 weeks is shown in FIG. 15.

Some noticeable changes in degradation products were observed at 40° C.,which is a stress condition for a liquid. Brief accelerated stability (2weeks at 40° C.) shows that formulations 4, 5 and 6 offer comparable orimproved stability compared to the control (10 mM histidine, 5% sucrose,0.01% polysorbate-80, pH 6.0). Formulations 1, 2 and 3 appear to have anegative impact on stability.

The data show that glycine, methionine, and increased sucrose arestabilizing to high concentration TNF-binding nanobody liquidformulations. The data show that arginine base, arginine hydrochlorideand sodium chloride may be de-stabilizing to high concentrationTNF-binding nanobody liquid formulations under some conditions.

Example 6 Stability of TNF-Binding Nanobody of High Concentration LiquidFormulation, Short-Term (2 Weeks Duration), Histidine and Tris Buffers

Stability of TNF-binding nanobody as a liquid is exemplified in thefollowing FIGS. 16-19. Two formulations were examined: ATN-103 at 118mg/mL in 20 mM Histidine, 5% Sucrose, pH 6.0; and ATN-103 at 117 mg/mLin 20 mM Tris, 5% Sucrose, pH 7.2. The stability of the formulations wasassessed by percentage of HMW and percentage of LMW by SE-HPLC, andpercentage of acidic and percentage of basic species by CEX-HPLC. Thedata show limited changes in degradation products as a function ofstorage time at 4° C. Some noticeable changes in degradation productswere observed at 40° C., which is a stress condition for a liquid. Thedata show that the stability of TNF-binding nanobody in histidine andtris buffers is essentially similar under these formulation conditions,with histidine performing slightly more favorably (slightly less LMW).Pre-formulation activities would later determine that the elevated pH (7or greater) results in a greater degree of LMW formation, explaining theadvantage observed below.

Example 7 Stability of High Concentration Liquid Formulation ofTNF-Binding Nanobody: Assessment of Interfacial Stresses (Freeze/Thaw)

FIGS. 20-23 demonstrate the stability of liquid TNF-binding nanobodyformulation at approximately 80 mg/mL in 10 mM Histidine, 5% Sucrose,0.01% Polysorbate 80, pH 6.0. Assessment was based on Sizeexclusion-HPLC, turbidity, and concentration assessment followingmultiple freeze-thaw cycling from −80° C. and 37° C.

The data show limited change in stability as a function of multiplefreeze-thaw cycling from −80° C. and 37° C.

Example 8 Stability of High Concentration Liquid Formulation ofTNF-Binding Nanobody: Assessment of Short-Term Thermal StressesPotentially Encountered in Manufacturing Processes

FIG. 24 demonstrates that liquid TNF-binding nanobody is robust toshort-term thermal stresses that might potentially be encountered duringdrug substance and drug product manufacturing processes. The highconcentration liquid was studied in 10 mM Histidine, 5% Sucrose, 0.01%Polysorbate 80, pH 6.0, at approximately 80 mg/mL and 50 mg/mL.Assessment was based on percentage of HMW and percentage of LMW by Sizeexclusion-HPLC, after exposure for 8 hours at 40° C., 7 days at 25° C.,and 29 days at 5° C. The data show limited changes in aggregates as afunction of storage time at 5° C. and 25° C. Some changes in aggregateswere observed at 40° C., which is a stress condition for a liquid.

The percentage of LMW species by SE-HPLC for TNF-binding nanobody highconcentration liquid was below limit of detection (i.e. 0.0%) at thetemperatures and durations indicated.

Example 9 Stability of Low Concentration Liquid Formulation of ATN-103:Assessment of Optimal pH and Formulation

FIGS. 25-28 demonstrate the stability of a liquid TNF-binding nanobodyformulations at low concentration (approximately 1 mg/mL) buffered at pH5.5, 6.0, 6.5, and 7.0. The stability of low concentration liquidTNF-binding nanobody was examined as a function of formulation and pH,in response to stress such as exposure to 40° C. temperature (FIGS. 25and 26), shaking (FIG. 28), and freeze/thaw. Four pH were evaluated foreach of the three following formulations: 10 mM histidine, 5% sucrose,0.01% Tween-80; 10 mM histidine, 5% sucrose, 0.01% Tween-80, 150 mMarginine HCl; and 10 mM histidine, 5% sucrose, 0.01% Tween-80, 75 mMsodium chloride. In this data set, Tween-80 is used as a synonym forPolysorbate-80. Study samples were evaluated using SE-HPLC and UV (forboth concentration and turbidity—measured by A455).

Figure Codes:

HST: 10 mM histidine, 5% sucrose, 0.01% Tween-80

HSTA: 10 mM histidine, 5% sucrose, 0.01% Tween-80, 150 mM arginine HCl

HSTS: 10 mM histidine, 5% sucrose, 0.01% Tween-80, 75 mM sodium chloride

Results show that pH range of 5.5±7.0 is suitable for the formulation.The data show that under some conditions, pH 7.0 may show somedetrimental effects (increased low molecular weight species). The datashow that there is no significant benefit in adding arginine HCl orsodium chloride to the drug substance formulation, and in some cases maybe destabilizing.

FIG. 27 shows the percentage of HMW species by SE-HPLC for TNF-bindingnanobody after storage at 4° C., where essentially no change wasobserved after 4 weeks. The percentage of LMW species by SE-HPLC forTNF-binding nanobody low concentration was below limit of detection(i.e. 0.0%) at 4° C. for all solution conditions tested. No significantchanges were observed in HMW or LMW species by SE-HPLC, or UV A280, orA455, as a result of multiple freeze-thaw cycles.

Example 10 Low Concentration TNF-Binding Nanobody Liquid: Assessment ofthe Effect of Shaking as a Function of pH and Formulation

Data is also presented to show that TNF-binding nanobody is sensitive toshaking at 300 rpm for 4 hours (at 15° C.) over this pH range (FIG. 28).Formulations containing sodium chloride and arginine are especiallysensitive to shaking. The histidine, sucrose, tween-80 formulationshowed the least high molecular weight degradation within each pH group.The histidine, sucrose, tween-80 formulation at pH 6.0 and 7.0 showedthe least HMW degradation.

The UV absorbance of low concentration TNF-binding nanobody aftershaking was monitored at 280 nm (to monitor concentration) and 455 nm(to monitor turbidity). No significant changes were observed as a resultof shaking.

Low concentration TNF-binding nanobody solutions were examined aftermultiple freeze-thaw cycles by SE-HPLC and UV analysis at 280 nm (tomonitor concentration) and 455 nm (to monitor turbidity). No significantchanges were observed in SE-HPLC or UV A280 or A455 as a result ofmultiple freeze-thaw cycles.

Example 11 Stability of TNF-Binding Nanobody of High ConcentrationLiquid Formulation, Short-Term (2 Weeks Duration), Examining TonicityAdjusting Agents

The stability of the TNF-binding nanobody as a liquid is exemplified inthe following:

Five formulations were examined as shown in FIGS. 31 and 32 referred toherein as HST, HSGT, HSGMT, HSorb and Control. Each of the formulationsexamined described below.

FIGS. 31 and 32 Formulations HST 100 mg/mL TNF-binding nanobody, 20 mMhistidine, 8% sucrose, 0.01% polysorbate 80 HSGT 100 mg/mL TNF-bindingnanobody, 20 mM histidine, 5% sucrose, 80 mM glycine, 0.01% polysorbate80 HSGMT 100 mg/mL TNF-binding nanobody, 20 mM histidine, 5% sucrose, 80mM glycine, 10 mM methionine, 0.01% polysorbate 80 HSorb 100 mg/mLTNF-binding nanobody, 20 mM histidine, 5% sorbitol Control 100 mg/mLTNF-binding nanobody, 20 mM histidine, 5% sucrose

The formulations were stored as a liquid for two weeks at 4° C. and 40°C. (stress condition), in polypropylene tubes and in cyclic olefincopolymer prefilled syringe with a rubber plunger.

The stability of the formulations was assessed by percentage of HMW andpercentage of LMW by SE-HPLC as depicted in FIGS. 31 and 32. The datashow limited changes in degradation products as a function of storagetime at 4° C. For the samples shown in FIG. 32, no LMW was detected atthe initial time point, or after two weeks at 4° C. LMW was onlydetected in the 40° C. (stressed) samples. The data show all fiveformulations show comparable changes in degradation products as afunction of storage time at the stress condition 40° C. Thus, the datashow that all formulations are suitable for liquid dosage form.

Example 12 Stability of TNF-Binding Nanobody at Low Concentration andHigh Concentration Liquid Formulation, Confirming Target Formulation,and Examining Primary Packaging Containers

Stability of TNF-binding nanobody as a liquid is exemplified in thefollowing: Three formulations were examined:

-   -   (a) 10 mg/mL TNF-binding nanobody, 20 mM histidine, 7.5%        sucrose, 0.01% polysorbate 80;    -   (b) 50 mg/mL TNF-binding nanobody, 20 mM histidine, 7.5%        sucrose, 0.01% polysorbate 80;    -   (c) 100 mg/mL TNF-binding nanobody, 20 mM histidine, 7.5%        sucrose, 0.01% polysorbate 80.

The formulation was prepared in the following primary packagingcontainers:

(a) prefillable Type I pharmaceutical grade glass syringe from onevendor and a West 4432 siliconized gray rubber plunger

(b) prefillable glass Type I pharmaceutical grade syringe from a secondvendor and a West 4432 siliconized gray rubber plunger

(c) prefillable cyclic olefin copolymer and a West 4432 siliconized grayrubber plunger

The formulations were analyzed at t=0 and were found to be satisfactory.The formulation has been stored at 4° C., 25° C. and 40° C. for threemonths.

EQUIVALENTS

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

1. A formulation comprising: (a) a TNF-binding nanobody molecule at a concentration from about 10 mg/mL to about 250 mg/mL; (b) a lyoprotectant chosen from sucrose, sorbitol, or trehalose at a concentration of about 5% to about 10%; (c) a surfactant chosen from polysorbate-80 or poloxamer-188 at a concentration of about 0.01% to 0.6%; and (d) a buffer chosen from Histidine buffer at a concentration about 10 to about 20 mM, or a Tris buffer at a concentration about 20 mM such that the pH of the formulation is about 5.0 to 7.5, wherein the TNF-binding nanobody molecule in the formulation retains at least about 70% of its binding activity after storage for at least three months at 4° C.
 2. The formulation of claim 1, which has: (i) less than 5% of high molecular weight (HMW) species after storage for at least 12 months at 4° C.; (ii) less than 5% of low molecular weight (LMW) species after storage for at least 12 months at 4° C.; (iii) less than 10% of acidic species after storage for at least 12 months at 4° C.; and/or (iv) less than 5% of basic species after storage for at least 12 months at 4° C.
 3. The formulation of claim 1 or 2, which is in liquid, lyophilized, reconstituted lyophilized, or frozen bulk storage form.
 4. The formulation of claim 2, which is a liquid or lyophilized formulation comprising: (a) a TNF-binding nanobody molecule at a concentration from about 10 mg/mL to about 130 mg/mL; (b) sucrose at a concentration of about 5% to about 10%; (c) polysorbate-80 at a concentration of about 0.01%-0.02%; and (d) a buffer selected from the group consisting of Histidine buffer at a concentration about 10 to about 20 mM, such that the pH of the formulation is about 5.0 to 7.5.
 5. The formulation of claim 2, which is a bulk storage formulation comprising: (a) a TNF-binding nanobody molecule at a concentration from about 80 mg/mL to about 280 mg/mL; (b) sucrose at a concentration of about 5% to about 10%, (c) polysorbate-80 at a concentration of about 0.01% to 0.02%; and (d) a buffer selected from the group consisting of Histidine buffer at a concentration about 10 to about 20 mM, such that the pH of the formulation is about 5.0 to 7.5, wherein at least 100 liters of the formulation are stored at below freezing conditions.
 6. The formulation of claim 2, wherein the pH of the formulation is selected from the group consisting of 5, 5.5, 5.8-6.1, 6.0, 6.1, 6.5 and
 7. 7. The formulation of claim 2, wherein the sucrose, sorbitol or trehalose is at a concentration of about 5%, about 7.5%, or about 10%.
 8. The formulation of claim 2, wherein the TNF-binding nanobody molecule is a single chain polypeptide comprised of one or more single domain molecules.
 9. The formulation of claim 8, wherein the TNF-binding nanobody molecule is monovalent or multivalent.
 10. The formulation of claim 8, wherein the TNF-binding nanobody molecule is monospecific or multispecific.
 11. The formulation of claim 8, wherein one or more single domain molecules is CDR-grafted, humanized, camelized, de-immunized, or selected by phage display.
 12. The formulation of claim 8, wherein the TNF-binding nanobody molecule is a single chain fusion polypeptide comprising one or more single domain molecules that binds to tumor necrosis factor α (TNF α), and one single domain molecule that binds to human serum albumin (HSA) protein.
 13. The formulation of claim 2, wherein the TNF-binding nanobody molecule comprises the amino acid sequence shown in FIG. 30 (SEQ ID NO:1), or an amino acid sequence at least 90% identical thereto.
 14. The formulation of claim 2, wherein at least one of the single domain molecules of the TNF-binding nanobody molecule comprises three CDRs having the amino sequence: DYWMY (SEQ ID NO:2) (CDR1), EINTNGLITKYPDSVKG (SEQ ID NO:3) (CDR2) and SPSGFN (SEQ ID NO:4) (CDR3), or having a CDR that differs by 1 conservative amino acid substitution from one of said CDRs.
 15. The formulation of claim 2, wherein at least one of the single domain molecules of the TNF-binding nanobody molecule comprises a variable region having the amino acid sequence from about amino acids 1 to 115 of FIG. 30 (SEQ ID NO:1), or a variable region that differs by up to 10 amino acids from said variable region.
 16. The formulation of claim 2, wherein the TNF-binding nanobody molecule further comprises at least one single domain molecule that binds to HSA and comprises three CDRs having the amino sequence: SFGMS (SEQ ID NO:5) (CDR1), SISGSGSDTLYADSVKG (SEQ ID NO:6) (CDR2) and GGSLSR (SEQ ID NO:7) (CDR3), or having a CDR that differs by 1 conservative amino acid substitution from one of said CDRs.
 17. The formulation of claim 2, wherein at least one of the single domain molecules of the TNF-binding nanobody molecule binds to HSA and comprises a variable region having the amino acid sequence from about amino acids 125 to 239 of FIG. 30 (SEQ ID NO:1), or a variable region that differs by up to 10 amino acids from said variable region.
 18. A method or process of preparing a formulation of a TNF-binding nanobody, comprising: expressing the TNF-binding nanobody in a cell culture; purifying the TNF-binding nanobody by passing the TNF-binding nanobody through at least one of a chromatography purification step, or an ultrafiltration/diafiltration steps; adjusting the concentration of the TNF-binding nanobody to about 10 to 250 mg/mL in a formulation containing sucrose at a concentration of about 5% to about 10%; polysorbate-80 at a concentration of about 0.01%, 0.02%; and a Histidine buffer at a concentration about 10 to about 20 mM or a Tris buffer at a concentration about 20 mM, such that the pH of the formulation is about 5 to 7.5.
 19. A method of preparing a reconstituted formulation containing a TNF-binding nanobody molecule, comprising: lyophilizing a mixture of a TNF-binding nanobody molecule and a lyoprotectant, a surfactant, and a buffer, thereby forming a lyophilized mixture; and reconstituting the lyophilized mixture in a diluent, thereby preparing the formulation, wherein the reconstituted formulation comprises (a) a TNF-binding nanobody molecule at a concentration of about 10 mg/mL to about 130 mg/mL; (b) a lyoprotectant selected from the group consisting of sucrose or trehalose at a concentration of about 5% to about 10%; (c) polysorbate-80 as the surfactant at a concentration of about 0.01% to 0.02%; and (d) a Histidine buffer at a concentration about 10 to about 20 mM, or a Tris buffer at a concentration about 20 mM, such that the pH of the formulation is about 5.0 to 7.5.
 20. A kit or an article of manufacture, comprising a container containing the formulation of any of claim 1, 2 or 13, and instructions for use.
 21. The kit or article of manufacture of claim 20, wherein the formulation is present in a vial or an injectable syringe.
 22. The kit or article of manufacture of claim 20, wherein the formulation is present in a prefilled injectable syringe.
 23. The kit or article of manufacture of claim 21, wherein the syringe or a vial is composed of glass, plastic, or a polymeric material chosen from a cyclic olefin polymer or copolymer.
 24. A method of treating or preventing a TNF-related disorder, comprising administering to a subject, a pharmaceutical composition that comprises the formulation of any of claim 1, 2 or 13, thereby reducing one or more symptoms associated with the TNF-related disorder.
 25. The method of claim 24, wherein the TNF-related disorder is an inflammatory or an autoimmune disorder.
 26. The method of claim 24, wherein the TNF-related disorder is chosen from rheumatoid arthritis (RA), arthritic conditions (e.g., psoriatic arthritis, polyarticular juvenile idiopathic arthritis (JIA), ankylosing spondylitis (AS), psoriasis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, or multiple sclerosis.
 27. A method of analyzing a manufacturing process, comprising: providing a sample of the formulation of claim 1, 2 or 13; assessing a parameter of the formulation chosen from color, clarity, viscosity, or an amount of one or more HMW, LMW, acidic or basic species; determining whether the parameter meets a preselected criteria, thereby analyzing the process.
 28. The method of claim 27, further comprising comparing two or more sample formulations in a method of monitoring or controlling batch-to-batch variation or to compare the sample to a reference standard.
 29. The method of claim 28, further comprising classifying, selecting, accepting or discarding, releasing or withholding, processing into a drug product, shipping, moving to a different location, formulating, labeling, packaging the formulation, based upon the comparison.
 30. The method of claim 29, further comprising providing a record which includes data relating to the assessed parameter of the formulation and optionally includes an identifier for a batch of the formulation; submitting said record to a decision-maker; optionally, receiving a communication from said decision maker; optionally, deciding whether to release or market the batch of formulation based on the communication from the decision maker. 